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(Received for publication, November 14, 1994) From the
The CD52 antigen was extracted from human spleens with organic
solvents and purified by immunoaffinity and reverse-phase
chromatography. The latter step resolved two CD52 species, called
CD52-I and CD52-II. Both species were found to contain similar N-linked oligosaccharides and glycosylphosphatidylinositol
(GPI) anchor glycans. The N-linked oligosaccharides were
characterized by methylation linkage analysis and, following exhaustive
neuraminidase and endo- The difference
between CD52-I and CD52-II was in the phosphatidylinositol (PI)
moieties of the GPI anchors. The phosphatidylinositol-specific
phospholipase C-sensitive CD52-I contained exclusively distearoyl-PI,
while the PI-phospholipase C-resistant CD52-II contained predominantly
a palmitoylated stearoyl-arachidonoyl-PI, as judged by electrospray
ionization mass spectrometry. Tandem mass spectrometric studies
indicated that the palmitoyl residue of the CD52-II anchor is attached
to the 2-position of the myo-inositol ring. Both the CD52-I
and CD52-II PI structures are unusual for GPI anchors and the possible
significance of this is discussed. The alkali-lability of the CD52
epitope recognized by the Campath-1H monoclonal antibody was studied.
The data suggest that the alkali-labile hydroxyester-linked fatty acids
of the GPI anchor are necessary for antibody binding.
The antigen recognized by CD52 antibodies, referred to herein as
CD52 and also known as the Campath-1 antigen, is a
glycosylphosphatidylinositol (GPI) ( The structure
of CD52 is unusual in that it is a very small, heavily glycosylated
molecule with glycolipid-like properties (Xia et al., 1991).
CD52 homologues have been described in mouse and rat (Kubota et
al., 1990; Kirchhoff, 1994). Furthermore, CD24 (Kay et
al., 1991), and its mouse homologue J11D (Kay et al.,
1990), appear to be quite similar to CD52. Previous work (Xia et
al., 1993a) has shown that mature CD52 contains a short peptide
(12 amino acids) linked to the membrane via a GPI anchor. It can be
separated into two distinct fractions (called herein CD52-I and
CD52-II) that differ in their hydrophobicity and susceptibility to
phosphatidylinositol-specific phospholipase C (PI-PLC). Both forms of
the antigen carry one N-linked oligosaccharide which is not
essential for antigenic activity. The epitope(s) recognized by the
anti-CD52 monoclonal antibodies Campath-1M and Campath-1H are labile to
alkaline conditions (Xia et al., 1993a) but the structural
basis for this lability is unknown. In this paper we describe the
complete primary structure of this clinically important molecule,
including the GPI anchor and N-linked oligosaccharide
moieties, and provide data on the nature of the alkali-labile epitope.
The
deaminated, reduced GPI-peptide was split into three aliquots (1, 2,
and 3) which were subjected to partial acid hydrolysis in 50 µl of
0.1 M trifluoroacetic acid (100 °C, 4 h) (Schneider and
Ferguson, 1995). Aliquot 2 was digested with 250 milliunits of jack
bean
Released and reduced N-linked glycans were digested
with A. ureafaciens sialidase (0.2 unit) in 200 µl of 100
mM sodium acetate, pH 5.0, at 37 °C for 18 h. Released,
reduced, and desialylated N-linked glycans were digested with
endo- CD52-I was digested with B. cereus phospholipase C in 25 µl of 25 mM Tris acetate, pH
7.4, 0.1% sodium deoxycholate for 24 h at 37 °C. Aliquots of 1.5
µl of enzyme suspension in 3.2 M ammonium sulfate were
added at 0 and 8 h. Control samples were incubated in parallel with
aliquots of 3.2 M ammonium sulfate.
The structures of CD52-I and CD52-II are shown in Fig. 1, together with a summary of the manipulations used in
this study. The only detectable difference between the two forms of the
antigen were in the phosphatidylinositol moiety.
Figure 1:
Structure of CD52-I and CD52-II and
summary of treatments. CD52-I and CD52-II differ only in the presence
(CD52-II) or absence (CD52-I) of a palmitoyl residue (R
Figure 2:
Microsequencing of the GPI neutral glycans
of CD52-I and CD52-II. Panel A, an authentic standard of
Man
Figure 3:
Positions
of the ethanolamine phosphate groups. Panel A, a partial
hydrolysate of deaminated and reduced CD52-II GPI-peptide was digested
with jack bean
Figure 4:
Electrospray mass spectrometric analysis
of the PI moieties of CD52-I and CD52-II. Panel A, negative-ion spectrum of the CD52-I PI fraction. Panel B,
negative ion spectrum of the CD52-II PI fraction. Panel C,
daughter ion spectrum of the m/z 1124 pseudomolecular ion of
CD52-II PI. Panel D, daughter ion spectrum of the m/z 885 pseudomolecular ion of bovine liver
1-stearoyl-2-arachidonoyl-PI. Panel E, fragmentation scheme
for the collision induced dissociation of
1-stearoyl-2-arachidonoyl-PIs. R
The m/z 866 ion from CD52-I can be interpreted as
the[M-1] The m/z 1124 ion from CD52-II can be
interpreted as the [M-1] The lipid moiety of
CD52-II is considerably more heterogeneous than that of CD52-I (Fig. 4B). Only the ion at m/z 1124 was
sufficiently intense to perform tandem mass spectrometry, however, the
other pseudomolecular ions have been tentatively assigned based on
their m/z values alone (Table 1).
The daughter ion
spectrum of the 1-stearoyl-2-arachidonoyl-PI standard (Fig. 4D) shows an intense fragment ion at m/z 241 that corresponds to inositol-1,2-cyclic phosphate (Sherman et al., 1985). This ion is absent from the corresponding
spectrum of the palmitoylated (stearoyl-arachidonoyl)-PI from CD52-II (Fig. 4C). This result strongly suggests that the
palmitoyl residue is esterified to the 2-position of the inositol ring,
and therefore prevents the formation of this ion. The interpretations
of the other fragment ions in Fig. 4, C and D,
are shown in Fig. 4E.
The GPI-peptide derived from
CD52-I was analyzed by positive-ion ESI-MS and produced the spectrum
shown in Fig. 5A. After transformation, these data
indicated the presence of a major molecular species of mass 2951.1
± 1.6 Da. The theoretical average mass of the CD52-I GPI-peptide
shown in Fig. 1is 2951.5 Da. The close agreement in the
measured and theoretical masses are consistent with the suggested
composition of the major CD52-I GPI-peptide component (i.e. the dodecapeptide sequence, the trimannosyl-glucosaminyl glycan
structure, the two ethanolamine phosphate groups, and the
distearoylphosphatidylinositol lipid moiety). Partial alkaline
hydrolysis of the CD52-I GPI-peptide prior to negative-ion ESI-MS gave
a spectrum containing two ions at m/z 1208.1 and 1341.1 that
can be interpreted as the [M-2H]
Figure 5:
Electrospray mass spectrometric analysis
of the GPI-peptide of CD52-I. Panel A, positive-ion spectrum
of the GPI-peptide of CD52-I. The ions at m/z 1475.8 (A2) and
984.4 (A3) correspond to the [M+2H]
The minor
molecular species of 2933.5 ± 0.8 Da (Fig. 5A)
is 17.6 ± 2.4 Da smaller than the major species. This difference
could be due to substitution of a Ser residue by Ala within the peptide
sequence (16 Da theoretical difference). The DNA codon for the
COOH-terminal Ser in the mature peptide is TCA (Xia et al.,
1991) which could be mutated to the Ala codon GCA by a single point
mutation. Although only one gene has been identified for CD52
(Kirchhoff et al., 1993) it is worth noting that the purified
antigen studied here was prepared from a pool of 12 whole spleens and
might therefore be subject to genetic polymorphism. The GPI-peptide of
CD52-II failed to give ESI mass spectra or matrix-assisted laser
desorption ionization mass spectra for reasons that are not clear.
Figure 6:
HPAEC
separation of the N-linked oligosaccharides of CD52-I and
CD52-II. N-Linked oligosaccharides were released by PNGase F
digestion and reduced with NaB
Exhaustive digestion of the desialylated oligosaccharide fractions
with endo-
Figure 7:
Endo-
The major core structure, corresponding to the 16.5 Gu peak of pool
(b), was sequenced using the reagent array analysis method
(RAAM
Figure 8:
RAAM
Figure 9:
Suggested structures of the N-linked oligosaccharides of CD52. Panel A,
structures of N-linked oligosaccharides based on the 16.5 core
structure (approximately 30% of the structures). The core structure is
represented by the shaded area. These structures could carry
one, two, or three polylactosamine chains. Panel B, structures
of N-linked oligosaccharides based on the 17.5 Gu core
structure (approximately 20% of the structures). The core structure is
represented by the shaded area. The square brackets indicate that substituents cannot be localized to a particular
branch. These structures could carry one or two polylactosamine chains. Panel C, structures of N-linked oligosaccharides
based on the four 23.0 Gu core structures (approximately 35% of the
structures). It is possible that these structures contain a linear
polylactosamine chain in addition to the branched polylactosamine
structure. The numbers of the polylactosamine repeats (x, y, and z) are unknown. Ambiguities in
linkage sites are indicated by square
brackets.
Pool (a), containing the unfractionated
23.0 Gu core structures, was subjected to complete RAAM
Figure 10:
Analysis of the Campath-1H epitope of
CD52-I using a WGA sandwich ELISA. CD52-I was hydrolyzed with 100
mM NaOH (dashed line), digested with PI-PLC (dotted line), or mock treated (solid line) and
adsorbed directly to plastic ELISA plates (panel A) or bound
to WGA-coated ELISA plates (panel B) and detected with
Campath-1H antibody. In panel B, + indicates the extent
of binding of CD52-I from PBS to the ELISA plate in the absence of
WGA.
The results (Fig. 10B) show that mock-treated CD52-I
binds well to the WGA-coated plate and that it can be detected with the
Campath-1H antibody. In contrast, PI-PLC-treated CD52-I, which
presumably still binds to the WGA, is no longer detected by the
Campath-1H antibody. Similar results were obtained using alkaline
hydrolysis (Fig. 10B). CD52 antigen is an unusual molecule with a very short peptide
element (12 amino acids) linked to a large sialylated,
polylactosamine-containing core-fucosylated tetraantennary N-linked oligosaccharide and to a simple GPI membrane anchor.
The major component of the molecule is, therefore, the large N-linked oligosaccharide. It is possible that this may be the
most important feature of the molecule with respect to possible
interactions with other molecules and/or cell surfaces. The molecule
behaves like a glycolipid, in terms of solvent solubility, which is
consistent with the deduced structure. The CD52 molecules can be
divided into two subclasses (CD52-I and CD52-II). Both subclasses
contain the same types of N-linked oligosaccharide and the
same GPI anchor carbohydrate structure, but differ in the PI moiety of
the GPI anchor (see Fig. 1). Studies using cloned cell lines
suggest that PI-PLC-sensitive GPI anchors (as found on CD52-I) and
PI-PLC-resistant anchors (as found on CD52-II) are often cell
type-specific (Toutant et al., 1990; Richier et al.,
1992; Wong and Low, 1994). Since the CD52 preparation studied here was
from whole human spleens, it is possible that the two CD52 subclasses
are due to the presence of multiple cell types expressing CD52 in this
organ. In general, the GPI PI moieties are substantially different
from the cellular pool of PI phospholipids (McConville and Ferguson,
1993). For example, several of the mammalian GPI anchors contain
exclusively alkylacyl-PIs (Roberts et al., 1988; Walter et
al., 1990; Redman et al., 1994) as opposed to sn-1-stearoyl-2-arachidonoyl-PI that is the predominant
cellular PI species in these organisms (Kerwin et al., 1994). In the case of CD52-I, the PI moiety is exclusively distearoyl-PI.
Diacyl-PI moieties are known in some higher eukaryote GPI anchors, for
example, in Torpedo acetylcholinesterase
(Bütikofer et al., 1990). However, the
only other example of a GPI anchor diacyl-PI that contains exclusively
one type of acyl chain is that of T.brucei variant
surface glycoprotein. The dimyristoyl-PI moiety of the variant surface
glycoprotein anchor is produced by a process of fatty acid remodelling
(Masterson et al., 1990), whereby the original heterogeneity
in the PI moiety (Doering et al., 1994) is removed by
sequentially replacing the sn-2 and sn-1 fatty acids
with myristate. While most mammalian GPI intermediates and precursors
contain alkylacyl-PIs, some contain diacyl-PIs (Puoti and Conzelmann,
1993; Singh et al., 1994). Thus it is possible that some kind
of analogous fatty acid remodelling may occur on the
diacyl-PI-containing GPI intermediates in the cells producing CD52-I. The PI moiety of the CD52-II GPI anchor, predominantly palmitoylated
stearoyl-arachidonoyl-PI, is unusual in that it is the first example of
a GPI PI moiety with a glycerolipid structure that is similar to the
cellular PI phospholipid pool. The presence of the palmitoyl residue
attached to the inositol, which is a GPI-specific PI modification,
rules out any possible contamination of the sample with cellular PI
phospholipids. The identification of this palmitoylated
stearoyl-arachidonoyl-PI species suggests that, at least in the cell
types expressing CD52-II, the GPI biosynthetic pathway might proceed
from conventional arachidonoyl-stearoyl-PI without any lipid
remodelling. Thus CD52-I and CD52-II appear to display two extremes
of PI processing in GPI biosynthesis. This is rather striking
considering that the CD52-I and CD52-II structures appear to be
identical in all other aspects. The simplest explanation would be a
difference in the available GPI precursors in different cell types,
however, the possibility that one form is converted to the other at the
cell surface cannot be formally excluded. The function of CD52 is
unknown and, consequently, the functional significance of having two
forms of CD52 that differ only in their lipid structure is obscure. The presence of a palmitoyl residue in ester linkage to the inositol
ring is known to correlate with resistance to bacterial PI-PLC (Roberts et al., 1988) and this modification was localized to the 2-
and/or 3-position of the inositol ring of a procyclic T.brucei GPI anchor (Ferguson, 1992b). The absence of the m/z 241 inositol-1,2-cyclic phosphate ion in the tandem ESI-MS
data presented here provides the first direct indication that this
modification is exclusively at the 2-position. The presence of a
substitution at the 2-position of the inositol ring would explain the
PI-PLC resistance of the palmitoylated anchors, since the bacterial
PI-PLC enzymes operate via nucleophilic attack of the phosphorus atom
by the hydroxyl group at the 2-position of the inositol ring (Volwerk et al., 1990). The carbohydrate components of the GPI
anchors of the CD52-I and CD52-II molecules are identical and conform
to the consensus structure of all GPI anchors (McConville and Ferguson,
1993). The only lymphocyte GPI structure that has been reported is that
of rat thymocyte Thy-1 (Homans et al., 1988). In that case
about 30% of the GPI glycans were substituted by GalNAc. This feature
appears to be absent in human CD52. The number and positions of the
ethanolamine phosphate groups on CD52-II was determined using a new
method (Schneider and Ferguson, 1994) involving the partial acid
hydrolysis of the deaminated, NaB Both CD52-I and CD52-II contain the
epitope recognized by Campath-1H antibody. In both cases the epitope is
destroyed by mild alkaline hydrolysis (Xia et al., 1993a). It
had been suggested previously that this might be due to an O-linked carbohydrate epitope (Xia et al., 1991;
Valentin et al., 1992). However, this possibility can be ruled
out by the ESI-MS data of the Campath-1H-reactive GPI-peptide prepared
from immunopurified CD52-I (see Fig. 5). The major GPI-peptide
species detected (measured mass 2951 Da) can be assigned to the CD52
dodecapeptide plus a distearoyl-GPI anchor without further
substituents. These data also rule out the possibility of any
alkali-labile substituents, other than the two ester-linked stearoyl
groups of the PI moiety. This raised the possibility that the
alkali-lability of the Campath-1H epitope might be due to abrogation of
its ability to bind to plastic, rather than chemical destruction of the
epitope. To test this we used a sandwich ELISA system, on wheat germ
agglutinin-coated plates, that captured native alkali-treated and
PI-PLC-treated CD52-I via its N-linked oligosaccharide. The
results indicated that the removal of the stearic acid groups (either
as free fatty acids by alkaline hydrolysis or as distearoylglycerol by
PI-PLC treatment) prevented subsequent recognition by the Campath-1H
antibody. It would appear, therefore, that the Campath-1H antibody
requires the hydroxyester-linked fatty acids on the glycerol backbone
of the PI moiety for efficient binding. This probably means that CD52
needs to be present as a multivalent aggregate, or micelle, to achieve
high affinity binding to the Campath-1H antibody. However, the lipid
dependence of glycolipid conformation (Nyholm and Pascher, 1993) and of
the binding of certain antiglycosphingolipid antibodies (Yoshino et
al., 1982; Kannagi et al., 1983; Kiarash et al.,
1994) has been described previously. Therefore, the possibility that
the ester-linked fatty acids play a direct role in the epitope cannot
be excluded.
Volume 270,
Number 11,
Issue of March 17, 1995 pp. 6088-6099
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-galactosidase digestion, by the reagent
array analysis method
. The results showed that the single
CD52 N-glycosylation site is occupied by large sialylated,
polylactosamine-containing, core-fucosylated tetraantennary
oligosaccharides. The locations of the phosphoryl substituents on the
GPI anchor glycan were determined using a new and sensitive method
based upon partial acid hydrolysis of the GPI glycan.
)anchored glycopeptide
which is abundantly expressed on virtually all human lymphocytes (Hale et al., 1983, 1990; Xia et al., 1993a). Monoclonal
antibodies against this antigen are remarkably potent effectors of
complement mediated lysis (Xia et al., 1993b) and have been
widely used in vivo and in vitro for the control of
graft versus host disease and for the prevention of bone
marrow transplant rejection (Hale and Waldmann, 1994).
Materials
Aluminium-backed high performance thin
layer chromatography sheets were from Merck; jack bean
-mannosidase, peptide N-glycanase F, Arthrobacter
ureafaciens sialidase, and endo--galactosidase were from
Boehringer Mannheim; Aspergillus phoenicis -mannosidase
and the reagent array analysis method RAAM kit were from
Oxford GlycoSystems; bovine liver phosphatidylinositol was from Sigma;
NaB
H
(10-15 Ci/mmol) was from DuPont NEN.
The anti-CD52 monoclonal antibody Campath-1H (Riechmann et
al., 1988) was obtained from The Wellcome Foundation. Commercial Bacillus cereus phosphatidylcholine phospholipase C
preparations are known to contain some phosphatidylinositol-specific
phospholipase C (PI-PLC) activity (Ferguson et al., 1985). In
this study a B. cereus phosphatidylcholine phospholipase C
preparation from Sigma was checked for PI-PLC activity (using
[H]myristate-labeled glycolipid A as a GPI
substrate) and used as a source of B. cereus PI-PLC. All other
reagents were of the highest purity commercially available.Antigen Purification
CD52-I and CD52-II were
purified from human spleen using chloroform/methanol extraction,
affinity chromatography, and octyl-Sepharose chromatography as
described (Xia et al., 1993a). Quantities of CD52 were
estimated from myo-inositol measurements and assuming 1 mol of myo-inositol/mol of CD52.Generation of the GPI Anchor Neutral Glycan
Fraction
CD52-I (2.5 nmol) and CD52-II (3 nmol) were subjected
to nitrous acid deamination followed by NaBH reduction as described (Ferguson, 1992a), freeze dried,
redissolved in 50 µl of water, and dialyzed against water using a
2000-Da cut-off membrane (Spectrapore). This material was subjected to
dephosphorylation with 50% aqueous HF followed by downward paper
chromatography in butanol, ethanol, water (4:1:0.8, v/v) (Ferguson,
1992a) to produce the GPI anchor neutral glycan fraction. These glycans
contain 2,5-anhydromannitol (AHM) at their reducing terminus. Authentic
standards of
Man1-2Man1-2Man1-6Man1-4AHM
(Man-AHM) and
Man1-2Man1-6Man1-4AHM
(Man-AHM) were prepared from the GPI anchors of yeast
glycoproteins and Trypanosoma brucei variant surface
glycoprotein (variant MITat1.5), respectively (Fankhauser et
al., 1993; Güther et al., 1992).Generation of the GPI-peptide and N-Glycan
Fractions
CD52-I (45 nmol) and CD52-II (56 nmol) were digested
with PNGase F (10 milliunits) in 320 µl of 250 mM sodium
phosphate buffer, pH 7.4, 10 mM EDTA for 16 h at 37 °C.
The reaction mixtures were adjusted to 5% 1-propanol and applied slowly
(4 ml/h) to an octyl-Sepharose column (10 
125 mm) that had been
equilibrated in 5% 1-propanol in 100 mM ammonium acetate. The
column was eluted (10 ml/h) with a linear gradient to 60% 1-propanol in
water over 80 ml. Fractions (1 ml) were collected and 1-µl aliquots
of each fraction were screened by ELISA for the presence of CD52
epitope using Campath-1H antibody. The immunoreactive
GPI-peptide-containing fractions were pooled and freeze dried to remove
the ammonium acetate. The released N-linked oligosaccharides
were recovered from the octyl-Sepharose column flow-through and
desalted by gel filtration on a Bio-Gel P4 column (10 200 mm),
where they eluted in the void volume.Generation of the Deaminated, Reduced GPI-peptide
Fraction and Partial Acid Hydrolysis
The GPI-peptide of CD52-II
(2 nmol) was deaminated with 60 µl of 50 mM sodium acetate
buffer, pH 4.0, containing 250 mM sodium nitrite (2 h, room
temperature). After deamination, 24 µl of 0.4 M boric acid
was added and the phosphatidylinositol was extracted three times with
85 µl of water-saturated 1-butanol. The aqueous phase was reduced
by the addition of 6 µl of 2 M NaOH and 5 µl of 36
mM NaBH in 100 mM NaOH (1.5
h, room temperature). The reduction was completed by the addition of 10
µl of 1 M NaB
H (3 h, room
temperature) and the products were neutralized with 1 M acetic
acid. The deaminated, reduced GPI-peptide was desalted by passage
through 0.2 ml of AG-50-X12 (H
) followed by rotary
evaporation to dryness, dried twice from 250 µl of 5% acetic acid
in methanol, twice from 250 µl of methanol (to remove boric acid),
and twice from 100 µl of toluene (to remove acetic acid). The
radiolabeled GPI-peptides were subjected to downward paper
chromatography on Whatman 3MM paper using 1-butanol, ethanol, water
(4:1:0.8, v/v) for 60 h. The paper strips were analyzed using a linear
analyzer (Rita, Raytest) and the material that had stayed at the origin
was eluted with water. To achieve further radiochemical purity this
eluate was chromatographed on a Sephadex G-25 column (400 10
mm, 3 ml/h), equilibrated with 100 mM ammonium acetate. The
deaminated, reduced GPI-peptide eluted in the void volume.-mannosidase for 24 h at 37 °C in 15 µl of 0.1 M sodium acetate buffer, pH 5.0, boiled for 5 min and dried.
Subsequently, all three aliquots were dephosphorylated using 50 µl
of 50% aqueous HF (60 h, 0 °C), neutralized with LiOH, desalted,
and dried as described (Ferguson, 1992a). Aliquot 3 was then subjected
to the same jack bean -mannosidase treatment as described above
and desalted by passage through a column of 0.2 ml of AG-50-X12
(H) over 0.2 ml of AG-3X4 (OH
) over
0.1 ml of QAE-Sephadex A-25 (OH).Radiolabeling of N-linked Oligosaccharides
PNGase
F released N-linked oligosaccharides of CD52-I (20 nmol) and
CD52-II (20 nmol) were incubated separately with 5 µl of 36 mM NaBH in 30 µl of 400 mM sodium borate buffer, pH 10.5, for 90 min, followed by the
addition of 65 µl of 1 M NaBH and
further incubation for 3 h. The reduction mixture was neutralized with
1 M acetic acid and desalted as described above for the
deaminated, reduced GPI-peptide (excluding the Sephadex G-25 gel
filtration step).Generation of the PI Fraction
The GPI-peptide of
CD52-II (2 nmol) and intact CD52-I (2 nmol) were deaminated as
described for the generation of the deaminated, reduced GPI-peptide.
The released phosphatidylinositol in the 1-butanol phase was dried
under a stream of N and redissolved in 100 µl of
methanol/chloroform (3:2, v/v) for electrospray mass spectrometric
analysis.Enzymatic and Chemical Cleavages
Acetolysis, A. phoenicis -mannosidase, and jack bean
-mannosidase digestions were performed as described (Ferguson,
1992a).-galactosidase (15 milliunits) in 15 µl of 50 mM sodium acetate buffer, pH 5.8, 0.2 mg/ml bovine serum albumin, 0.5
mg/ml sodium azide for 40 h at 37 °C. After digestion the samples
were boiled for 5 min and desalted by passage through a column of 0.2
ml of AG-50-X12 (H) over 0.2 ml of AG-3X4
(OH) over 0.1 ml of QAE-Sephadex A-25
(OH).Alkaline Hydrolysis of CD52
CD52-I was subjected
to alkaline hydrolysis in 25 µl of 100 mM NaOH at 37
°C for 18 h, followed by neutralization with 25 µl of 100
mM HCl, and to partial alkaline hydrolysis in 50 µl of 20%
NH in 20% 1-propanol at 37 °C for 16 h, followed by
drying under a stream of N.ELISA
The basic ELISA for the detection of CD52
was performed as described previously (Xia et al., 1993a). For
the modified sandwich ELISA, Dynatech Immunolon 4 plates were coated
with wheat germ agglutinin (WGA) using 100 µg/ml WGA in
phosphate-buffered saline (PBS), pH 7.2, 4 °C, 16 h. The coated
plates were washed five times with 0.05% Tween 20 in Tris-buffered
saline, pH 7.2, and subsequently incubated with doubling dilutions of
CD52 samples in PBS for 2 h at room temperature. The plates were washed
five times with 0.05% Tween 20 in Tris-buffered saline and blocked with
2% (w/v) bovine serum albumin in PBS for 1 h at room temperature. After
incubation with Campath-1H antibody (5 µg/ml in PBS, 2% (w/v)
bovine serum albumin, 1.5 h, room temperature), and five washes, bound
CD52-Campath-1H complexes were detected with an alkaline
phosphatase-conjugated goat anti-human IgG 
-chain second antibody
(Sigma) (diluted 1:200 in PBS, 2% (w/v) bovine serum albumin, 1 h, room
temperature). After seven washes, the wells were developed with 100
µl of 2 mg/ml p-nitrophenyl phosphate in 1 M ethanolamine-HCl buffer, pH 9.5, 30 min, room temperature. The
reaction was stopped with 50 µl of 2 M NaOH and plates
were read at 405 nm using an Anthos automated ELISA plate reader.Composition and Methylation Analyses
Neutral
sugars, lipids, and inositol contents and methylation linkage analyses
were performed using gas chromatography-mass spectrometry, as described
previously (Ferguson, 1992a).High Performance Anion Exchange Chromatography
(HPAEC)
HPAEC was performed on a Dionex Bio-LC system fitted
with a Dionex PA-1 Carbopac column, a pulsed amperometric detector,
anion suppressor recycling system, and a radioactivity flow monitor
(Ramona, Raytest). The following high performance liquid chromatography
conditions were used: Program 1 (Ferguson, 1992a), flow rate 0.6
ml/min, linear gradient from 12.5 to 50 mM sodium acetate in
150 mM NaOH over 50 min. The chromatographic properties of the
neutral oligosaccharides were expressed in Dionex units (Du) by linear
interpolation of their elution positions between adjacent glucose
oligomer internal standards (dextran partial acid hydrolysate)
co-injected with the sample. The column was washed after each use with
250 mM sodium acetate in 150 mM NaOH. Program 2 (a
modified version of the method of (Anumula and Taylor, 1991)), flow
rate 1 ml/min, 8-min isocratic elution with 16 mM sodium
acetate in 180 mM NaOH followed by a linear gradient from 16
to 200 mM sodium acetate in 180 mM NaOH over 57 min.
The column was washed after each use with 250 mM sodium
acetate in 180 mM NaOH. Program 3 (a modified version of the
method of (Anumula and Taylor, 1991)), flow rate 0.6 ml/min, 8-min
isocratic elution with 20 mM sodium acetate in 100 mM NaOH followed by a linear gradient from 20 to 200 mM sodium acetate in 100 mM NaOH over 62 min. The column was
washed after each use with 200 mM sodium acetate in 100 mM NaOH. Program 4, flow rate 0.6 ml/min, 8-min isocratic at 20
mM sodium acetate in 100 mM NaOH followed by a linear
gradient from 20 to 75 mM sodium acetate in 100 mM NaOH over 82 min. The column was washed after each use with 200
mM sodium acetate in 100 mM NaOH.Thin Layer Chromatography of Labeled Glycans
GPI
anchor neutral glycans and reduced, desialylated N-linked
oligosaccharides, were chromatographed on Silica Gel 60 high
performance thin layer chromatography (HPTLC) sheets (Schneider et
al., 1993) using either solvent system 1: 1-propanol, acetone,
water (9:6:5, v/v) for the first and third developments; and propanol,
acetone, water (5:4:1, v/v) for the second development; solvent system
2: 1-butanol, ethanol, water (4:3:3, v/v), three or four developments;
or solvent system 3: 1-propanol, acetone, water (9:6:4, v/v) for one
development. Labeled glycans were detected by fluorography after the
sheets were sprayed with ENHANCE spray (DuPont NEN).Gel Filtration
Bio-Gel P4 chromatography was
performed using an Oxford GlycoSystems Glycosequencer in the high
resolution flow profile mode. Samples were co-injected with a dextran
partial acid hydrolysate. The data were analyzed with the software
provided by Oxford GlycoSystems.Reagent Array Analysis Method
(RAAM
The RAAM) sequencing of
NaBH-labeled N-linked oligosaccharides
(Edge et al., 1992) was carried out according to the
manufacturers' instructions. Briefly, a radiolabeled N-linked oligosaccharide is divided into nine aliquots which
are separately digested with various combinations of five different
exoglycosidases. After digestion (16 h at 37 °C) the products are
combined, desalted, and analyzed by gel filtration on a Bio-Gel P4
column. The elution profile is then compared to theoretical elution
profiles generated by the software. Alternatively, the nine digestion
products were individually desalted, applied to a Silica Gel 60 HPTLC
sheet, and developed with solvent system 2.Electrospray Ionization Mass Spectrometry
(ESI-MS)
ESI mass spectra were recorded on a VG Quattro
triple-quadrupole mass spectrometer (Fisons Instruments, VG-Biotech,
Altrincham, United Kingdom). Samples (10-100 pmol/µl) were
introduced into the electrospray source at 10 µl/min using a
Michrom high performance liquid chromatography pump. For positive-ion
mode mass spectrometry of the CD52-I GPI-peptide,
acetonitrile/water/formic acid (50:50:0.1, v/v/v) was used as a
solvent; for negative-ion mode mass spectrometry of partially base
hydrolyzed CD52-I GPI-peptide, 2-propanol/water (1:1, v/v) was used;
and for the negative-ion mode spectrometry of phosphatidylinositols,
methanol/chloroform (3:2, v/v) was used. For all tandem mass
spectrometric experiments the pseudomolecular parent ions were
accelerated into a collision cell containing argon (2.3
10
millibar) through a potential difference of
between 70 and 110 V. All the mass spectra were background subtracted
and smoothed using MassLynx software.
) on
position 2 of the myo-inositol ring and in the nature of the
R and R acyl/alkyl chains. R and
R are exclusively stearoyl residues in CD52-I, whereas they
predominantly are arachidonoyl and stearoyl residues in CD52-II (see Table 3). The following abbreviations are used: EtNH: ethanolamine; PNGase F, peptide-N-glycanase F; BuOH, 1-butanol; APAM, A. phoenicis (Man1-2Man
specific) -mannosidase; Ac
O, partial acetolysis; JBAM, jack bean -mannosidase. The alkali-labile bonds are
indicated on the structure of the GPI-peptide. The branched structure
attached to Asn-3 represents the N-linked
oligosaccharide.
Structure of the Glycosylphosphatidylinositol Membrane Anchor
Structure of the GPI Neutral Glycans
The neutral
glycans of the GPI anchors of CD52-I and CD52-II were isolated
following nitrous acid deamination, reduction with
NaBH, and aqueous HF dephosphorylation. The
radiolabeled glycans were purified by paper chromatography and
separated by Dionex HPAEC (Program 1). The chromatographic profiles for
CD52-I and CD52-II GPI neutral glycans were identical and contained a
major glycan peak at 2.4 Du (representing >95% of the neutral
glycans) and a minor glycan peak at 3.0 Du (data not shown). The 2.4-
and 3.0-Du glycan peaks from Dionex HPAEC were analyzed by Bio-Gel P4
chromatography where they displayed hydrodynamic volumes of 4.2 glucose
units (Gu) and 5.3 Gu, respectively (data not shown). These
chromatographic properties are characteristic of the structures
Man1-2Man1-6Man1-4AHM and
Man1-2Man1-2Man1-6Man1-4AHM,
respectively (Ferguson, 1992a). The putative sequence of the major
glycan was confirmed by HPTLC analysis (Schneider et al.,
1993) before and after Aspergillus phoenicis -mannosidase
digestion, acetolysis, and jack bean -mannosidase digestion (Fig. 2A). The minor glycan was shown to be sensitive
to jack bean -mannosidase, yielding exclusively AHM (data not
shown). Partial acid hydrolysis of this minor glycan produced a ladder
of structures (Fig. 2B) consistent with the
Man1-2Man1-2Man1-6Man1-4AHM
structure suggested from the Dionex HPAEC and Bio-Gel P4
chromatographic data.
1-2Man1-6Man1-4AHM
(Man-AHM) and the 2.4 Du neutral glycans from CD52-I and
CD52-II were subjected to A. phoenicis (Man1-2Man specific) -mannosidase (APAM)
digestion, partial acetolysis (AcO), and jack bean
-mannosidase (JBAM) digestion as indicated. Panel
B, the 3.0 Du neutral glycan from CD52-I was partially hydrolyzed
using trifluoroacetic acid (lane 2). The largest structure
co-chromatographs with an authentic
Man1-2Man1-2Man1-6Man1-4AHM
(Man-AHM) standard (lane 3) while the smallest
structure co-chromatographs with AHM (lane 1). The right-hand lane on both panels (Dex) is a reduced
dextran hydrolysate. HPTLC was performed using solvent system
1.
Positions of the Ethanolamine Phosphate Groups
The
positions of the phosphoryl substituents (ethanolamine phosphate
moieties) were assessed using a partial acid hydrolysate of the
deaminated and reduced GPI-peptide fraction of CD52-II. This material
was digested with jack bean -mannosidase before and after aqueous
HF dephosphorylation (Fig. 3A). This procedure exploits
the acid stability of the phosphoryl substituents and the resistance of
substituted -Man residues to -mannosidase digestion
(Schneider and Ferguson, 1995) (see Fig. 3B). The
individual bands in Fig. 3A, lanes 1 and 2, were quantitated and the results suggest that 100% of the
structures contain a phosphoryl substituent on the third (nonreducing
terminal) Man residue, approximately 90% of the structures contain a
phosphoryl substituent on the first Man residue (adjacent to the GlcN
residue) and approximately 40% contain a phosphoryl substituent on the
middle Man residue.
-mannosidase before (lane 2) and after (lane 3) dephosphorylation with aqueous HF. Lane 1 shows the dephosphorylated hydrolysate, the right-hand lane (Dex) is a reduced dextran hydrolysate. HPTLC was
performed using solvent system 3. Panel B, a schematic
representation of the sequence of reactions employed for the experiment
in panel A. The following symbols were employed: EtN,
ethanolamine; circled P, phosphate; 
, mannose; &cjs2108;,
glucosamine; &cjs0485;, myo-inositol; 
,
2,5-anhydromannitol; squiggley line, fatty
acid.
Isolation and Mass Spectrometric Analysis of the PI
Moieties
Intact CD52-I and the GPI-peptide of CD52-II were deaminated
to release their PI moieties which were recovered by solvent
extraction. Analysis of these fractions by negative ion ESI-MS revealed
major pseudomolecular ions at m/z 866 for CD52-I and m/z 1124 for CD52-II (Fig. 4, A and B).
is H in the case of bovine
liver PI and CH-(CH)
-CO
(palmitoyl) in the case of CD52-II PI. R is
CH-[(CH=CH)(CH)
]-
(for the arachidonoyl group) and R is
CH-(CH)
(for the stearoyl
group).
ion of a
distearoylphosphatidylinositol, an assignment which is consistent with
the compositional data for this molecule (Xia et al., 1993a)
and with the ESI mass spectral data described below for the
GPI-peptide. ion of a
palmitoylated (stearoyl-arachidonoyl)-phosphatidylinositol. This
assignment was confirmed by negative ion tandem mass spectrometry. The
daughter ion spectrum of m/z 1124 is shown in Fig. 4C and a daughter ion spectrum of authentic
1-stearoyl-2-arachidonoyl-PI is shown in Fig. 4D for
comparison. In these spectra, the two major fragment ions at m/z 283 and 303 correspond to the carboxylate ions of stearic acid and
arachidonic acid, respectively. The presence of a palmitoyl group in
the parent ion of m/z 1124 can be inferred by the presence of
the carboxylate fragment ion at m/z 255 (Fig. 4C). Interestingly, the intensity of the
palmitate ion is weaker than those of the stearate and arachidonate
ions. It has been noted in another study, on T. brucei procyclic acidic repetitive protein, ()that fatty acid
residues attached to the inositol ring produce weaker carboxylate
fragment ions than those attached to the glycerol backbone. Thus the
collision spectrum suggests that the palmitoyl component of CD52-II is
predominantly linked to the inositol ring.
Isolation and Mass Spectrometric Analysis of the
GPI-peptide
Attempts to obtain ESI mass spectra and matrix-assisted laser
desorption ionization mass spectra of native CD52-I and CD52-II were
unsuccessful. The extensive microheterogeneity of the N-linked
oligosaccharides of CD-52 (see below) was the most likely reasons for
this. The N-linked oligosaccharides were removed by digestion
with PNGase-F and the GPI-peptides were recovered by octyl-Sepharose
chromatography. The released N-linked oligosaccharides were
recovered in the flow-through for subsequent analysis (see below).
Carbohydrate analysis (Table 2) of the flow-through and
GPI-peptide fractions showed that the majority of the galactose and
fucose content of the starting material was found in the flow-through,
confirming that the removal of N-linked oligosaccharides by
PNGase-F was essentially complete.
pseudomolecular ions of the GPI-peptide minus 1 and 2 stearic
acid residues, respectively (Fig. 5B).
and the [M+H]
pseudomolecular ions of a molecule with a molecular mass of
2951.1 ± 1.6 Da. The peak at m/z 1468.2 corresponds to
the [M+2H] pseudomolecular ion of a
molecule with an molecular mass of 2933.5 ± 0.8 Da. Panel
B, negative-ion spectrum of the GPI peptide of CD52-I after
partial alkaline hydrolysis. The ions at m/z 1341.1 and 1208.1
correspond to the [M-2H] pseudomolecular ions of the GPI-peptide minus 1 and 2 stearoyl
residues (calculated masses 2682.2 and 2684.2 Da), respectively.
Analysis of the N-linked Oligosaccharides
Carbohydrate analysis of the released N-linked
oligosaccharides of CD52-I and CD52-II (Table 2) suggested the
presence of complex structures rich in Gal and GlcNAc and containing
Fuc and sialic acid. The H NMR spectra of the two fractions
were similar and suggested the presence of predominantly tetraantennary
structures containing mostly 2-6-linked sialic acid, with
some 2-3-linked sialic acid (data not shown). The
oligosaccharide fractions were reduced with NaBH and analyzed by Dionex HPAEC before and after desialylation with A. ureafaciens neuraminidase (Fig. 6, A and B). The elution profiles of the desialylated
oligosaccharide fractions suggested that they both contained very large
and highly heterogeneous structures (Anumula and Taylor, 1991).
H. Samples were
analyzed by HPAEC before (filled circles) and after (open
circles) digestion with A. ureafaciens neuraminidase. The sodium acetate gradients used are indicated in
the graphs with a solid line. Panel A,N-linked
oligosaccharides from CD52-I (Dionex HPAEC program 2). Panel B,N-linked oligosaccharides from CD52-II (Dionex HPAEC
program 3).
-galactosidase generated smaller core structures that
could be resolved by HPTLC (Fig. 7A) and Bio-Gel P4 gel
filtration (Fig. 7, B and C). The
endo--galactosidase digests were essentially identical, indicating
that the core structures are the same for CD52-I and CD52-II. The
endo--galactosidase digestion products from Fig. 7C were pooled as shown and rechromatographed by Dionex HPAEC. Pool
(a) was resolved into 4 structures and pool (b) was resolved into 2
major structures (16.5 and 17.5 Gu), data not shown. These data
indicate that the N-linked oligosaccharides of CD52-I and
CD52-II are highly heterogeneous and contain polylactosamine termini.
-galactosidase digestion of the N-linked oligosaccharides. Panel A, HPTLC analysis
(using solvent system 2) of CD52-I and CD52-II PNGase F, released
NaBH reduced desialylated N-linked
oligosaccharides before(-) and after (+)
endo--galactosidase (EG) digestion. The right-hand lane (Dex) is a reduced dextran
hydrolysate. Panel B, Bio-Gel P4 analysis of CD52-I N-linked oligosaccharides after endo--galactosidase
digestion. Panel C, Bio-Gel P4 analysis of CD52-II N-linked oligosaccharides after endo--galactosidase
digestion. The pooled fractions (a) and (b) are indicated by solid
bars. The numbers at the top of panels B and C represent the elution positions of glucose oligomer
internal standards (Gu values).
) (Edge et al., 1992). The result (Fig. 8A) gave a RAAM signature (Fig. 8B) consistent with four possible
core-fucosylated tetraantennary structures, two of which contained a
bisected outer-arm Man residue. The ambiguities in the result were
resolved by methylation analysis of the total N-linked
oligosaccharide fraction (Table 3). The absence of bisected
structures was indicated by the absence of a tri-O-substituted
Man residue, whereas the presence of di-O-substituted Man
residues indicates that the structure shown in Fig. 8C is the only feasible isomer. The 17.5 Gu core structure was
subjected to the same enzyme digestions used for the RAAM analysis. In this case, due to lack of material, the individual
digests were analyzed by HPTLC rather than Bio-Gel P4 (Table 4).
The data were consistent with the same structure as the 16.5 Gu
component plus another terminal Gal residue. Taking into account
the specificity of endo--galactosidase, the 16.5 Gu core must have
been originally substituted by one, two, or three linear
polylactosamine repeats (terminating in sialic acid) and the 17.5 Gu
core must have been originally substituted by one or two
polylactosamine repeats (terminating in sialic acid), Fig. 9, A and B.
analysis of the major
(16.5 Gu) core structure of the CD52-II N-linked
oligosaccharides. Panel A, Bio-Gel P4 analysis of the pooled
RAAM digests. Panel B, comparison of the
RAAM experimental signature with the best-matching
computer-generated theoretical signature. Panel C, suggested
structure of the 16.5 Gu N-linked
oligosaccharide.
analysis. However, the results were not immediately interpretable
because of the heterogeneity of structures in this peak. Nevertheless,
some information could be derived from the experimental signature, in
particular it was clear that all of the structures present must contain
a core -Fuc residue (data not shown). The four individual 23.0-Gu
species resolved from pool (a) by Dionex HPAEC were analyzed by
RAAM enzyme digestions and HPTLC, as described above for
the 17.5 Gu component (Table 4). Although the structures of the
23.0 Gu cores cannot be unambiguously assigned from these data, it
seems likely, given that they are the products of exhaustive
endo--galactosidase digestion, that the original oligosaccharides
terminate in branched structures similar to those shown in Fig. 9C.Sandwich ELISA Study of the Campath-1 Antibody Epitope
Native CD52 binds efficiently to plastic ELISA plates (Xia et al., 1993a). However, it was not clear from previous
studies whether the loss in CD52 antigenicity following PI-PLC
treatment or alkaline hydrolysis (Fig. 10A) was due to
destruction of the epitope itself, or simply to the abrogation of its
ability to bind to plastic in the absence of the PI-PLC/alkali-labile
lipid moiety. To address this issue a sandwich ELISA system was used
whereby CD52-I was captured via its N-linked oligosaccharide
on a surface of immobilized WGA rather than via its lipid moiety.
H-reduced
GPI-peptide (see Fig. 3). The results show that this is a
sensitive and reasonably quantitative method and that CD52-II, like
human erythrocyte acetylcholinesterase and bovine liver 5`-nucleotidase
(Deeg et al., 1992; Taguchi et al., 1994), contains
some structures with an ethanolamine phosphate group on each of the 3
conserved Man residues.
), reagent array analysis method; ESI-MS, electrospray
ionization mass spectrometry; Du, dionex units; Gu, glucose units;
PNGase F, protein N-glycanase F.)
We thank Brian N. Green (VG Biotech, Altrincham) for
help with the acquisition of the CD52-I GPI-peptide electrospray data.
We are grateful to Dr. Geoffrey Hale for helpful discussions.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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I. A. Brewis, M. A. J. Ferguson, A. Mehlert, A. J. Turner, and N. M. Hooper Structures of the Glycosyl-phosphatidylinositol Anchors of Porcine and Human Renal Membrane Dipeptidase J. Biol. Chem., September 29, 1995; 270(39): 22946 - 22956. [Abstract] [Full Text] [PDF]
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