(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--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.
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) ()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).
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 -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
).
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--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
).
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) 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 (Man
1-2Man
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.
Figure 2:
Microsequencing of the GPI neutral glycans
of CD52-I and CD52-II. Panel A, an authentic standard of
Man1-2Man
1-6Man
1-4AHM
(Man
-AHM) and the 2.4 Du neutral glycans from CD52-I and
CD52-II were subjected to A. phoenicis (Man
1-2Man specific)
-mannosidase (APAM)
digestion, partial acetolysis (Ac
O), 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
Man
1-2Man
1-2Man
1-6Man
1-4AHM
(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.
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 -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.
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 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).
The m/z 866 ion from CD52-I can be interpreted as
the[M-1] 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.
The m/z 1124 ion from CD52-II can be
interpreted as the [M-1] 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.
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] pseudomolecular ions of the GPI-peptide minus 1 and 2 stearic
acid residues, respectively (Fig. 5B).
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] 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.
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 NaBH
. 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).
Exhaustive digestion of the desialylated oligosaccharide fractions
with endo--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.
Figure 7:
Endo--galactosidase digestion of the N-linked oligosaccharides. Panel A, HPTLC analysis
(using solvent system 2) of CD52-I and CD52-II PNGase F, released
NaB
H
reduced desialylated N-linked
oligosaccharides before(-) and after (+)
endo-
-galactosidase (E
G) 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).
The major core structure, corresponding to the 16.5 Gu peak of pool
(b), was sequenced using the reagent array analysis method
(RAAM) (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.
Figure 8:
RAAM 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.
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 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.
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, NaBH
-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.
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.