©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Primary Structure of CD52 (*)

(Received for publication, November 14, 1994)

Achim Treumann M. Robert Lifely (1) Pascal Schneider Michael A. J. Ferguson (§)

From the Department of Biochemistry, University of Dundee, Dundee DD1 4HN, Scotland Department of Cell Biology, The Wellcome Foundation Research Laboratories, Beckenham, Kent BR33BS, United Kingdom

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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-beta-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.


INTRODUCTION

The antigen recognized by CD52 antibodies, referred to herein as CD52 and also known as the Campath-1 antigen, is a glycosylphosphatidylinositol (GPI) (^1)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.


EXPERIMENTAL PROCEDURES

Materials

Aluminium-backed high performance thin layer chromatography sheets were from Merck; jack bean alpha-mannosidase, peptide N-glycanase F, Arthrobacter ureafaciens sialidase, and endo-beta-galactosidase were from Boehringer Mannheim; Aspergillus phoenicis alpha-mannosidase and the reagent array analysis method RAAM kit were from Oxford GlycoSystems; bovine liver phosphatidylinositol was from Sigma; NaB^3H(4) (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 [^3H]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 NaB^3H(4) 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 Manalpha1-2Manalpha1-2Manalpha1-6Manalpha1-4AHM (Man(4)-AHM) and Manalpha1-2Manalpha1-6Manalpha1-4AHM (Man(3)-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 times 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 times 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 NaB^3H(4) in 100 mM NaOH (1.5 h, room temperature). The reduction was completed by the addition of 10 µl of 1 M NaB^2H(4) (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 times 10 mm, 3 ml/h), equilibrated with 100 mM ammonium acetate. The deaminated, reduced GPI-peptide eluted in the void volume.

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 alpha-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 alpha-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 NaB^3H(4) 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 NaB^2H(4) 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(2) and redissolved in 100 µl of methanol/chloroform (3:2, v/v) for electrospray mass spectrometric analysis.

Enzymatic and Chemical Cleavages

Acetolysis, A. phoenicis alpha-mannosidase, and jack bean alpha-mannosidase digestions were performed as described (Ferguson, 1992a).

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-beta-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.

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(3) in 20% 1-propanol at 37 °C for 16 h, followed by drying under a stream of N(2).

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 EN^3HANCE 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 NaB^3H(4)-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 times 10 millibar) through a potential difference of between 70 and 110 V. All the mass spectra were background subtracted and smoothed using MassLynx software.


RESULTS

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(3)) on position 2 of the myo-inositol ring and in the nature of the R(1) and R(2) acyl/alkyl chains. R(1) and R(2) 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(2): ethanolamine; PNGase F, peptide-N-glycanase F; BuOH, 1-butanol; APAM, A. phoenicis (Manalpha1-2Man specific) alpha-mannosidase; AcO, partial acetolysis; JBAM, jack bean alpha-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 NaB^3H(4), 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 Manalpha1-2Manalpha1-6Manalpha1-4AHM and Manalpha1-2Manalpha1-2Manalpha1-6Manalpha1-4AHM, respectively (Ferguson, 1992a). The putative sequence of the major glycan was confirmed by HPTLC analysis (Schneider et al., 1993) before and after Aspergillus phoenicis alpha-mannosidase digestion, acetolysis, and jack bean alpha-mannosidase digestion (Fig. 2A). The minor glycan was shown to be sensitive to jack bean alpha-mannosidase, yielding exclusively AHM (data not shown). Partial acid hydrolysis of this minor glycan produced a ladder of structures (Fig. 2B) consistent with the Manalpha1-2Manalpha1-2Manalpha1-6Manalpha1-4AHM structure suggested from the Dionex HPAEC and Bio-Gel P4 chromatographic data.


Figure 2: Microsequencing of the GPI neutral glycans of CD52-I and CD52-II. Panel A, an authentic standard of Manalpha1-2Manalpha1-6Manalpha1-4AHM (Man(3)-AHM) and the 2.4 Du neutral glycans from CD52-I and CD52-II were subjected to A. phoenicis (Manalpha1-2Man specific) alpha-mannosidase (APAM) digestion, partial acetolysis (Ac(2)O), and jack bean alpha-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 Manalpha1-2Manalpha1-2Manalpha1-6Manalpha1-4AHM (Man(4)-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 alpha-mannosidase before and after aqueous HF dephosphorylation (Fig. 3A). This procedure exploits the acid stability of the phosphoryl substituents and the resistance of substituted alpha-Man residues to alpha-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.


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 alpha-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; circle, 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).


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(3) is H in the case of bovine liver PI and CH(3)-(CH(2))(14)-CO (palmitoyl) in the case of CD52-II PI. R(1) is CH(3)-[(CH=CH)(4)(CH(2))(9)]- (for the arachidonoyl group) and R(2) is CH(3)-(CH(2)) (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, (^2)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.

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.



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+^3H] 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.

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 ^1H NMR spectra of the two fractions were similar and suggested the presence of predominantly tetraantennary structures containing mostly alpha2-6-linked sialic acid, with some alpha2-3-linked sialic acid (data not shown). The oligosaccharide fractions were reduced with NaB^3H(4) 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).


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^3H(4). 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-beta-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-beta-galactosidase digests were essentially identical, indicating that the core structures are the same for CD52-I and CD52-II. The endo-beta-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-beta-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^3H(4) reduced desialylated N-linked oligosaccharides before(-) and after (+) endo-beta-galactosidase (EbetaG) 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-beta-galactosidase digestion. Panel C, Bio-Gel P4 analysis of CD52-II N-linked oligosaccharides after endo-beta-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 betaGal residue. Taking into account the specificity of endo-beta-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 alpha-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-beta-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.


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).


DISCUSSION

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^3H(4)-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 alphaMan 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.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Howard Hughes International Research Scholar. To whom correspondence should be addressed. Tel.: 0382-229595; Fax: 0382-322583.

(^1)
The abbreviations used are: GPI, glycosylphosphatidylinositol; AHM, 2,5-anhydromannitol; PI-PLC, phosphatidylinositol-specific phospholipase C; ELISA, enzyme-linked immunosorbent assay; WGA, wheat germ agglutinin; PBS, phosphate-buffered saline; HPAEC, high performance anion exchange chromatography; HPTLC, high performance thin layer chromatography; RAAM, reagent array analysis method; ESI-MS, electrospray ionization mass spectrometry; Du, dionex units; Gu, glucose units; PNGase F, protein N-glycanase F.

(^2)
A. Treumann and M. A. J. Ferguson, unpublished data.


ACKNOWLEDGEMENTS

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.


REFERENCES

  1. Anumula, K. R., and Taylor, P. B. (1991) Eur. J. Biochem. 195, 269-280 [Abstract]
  2. Bütikofer, P., Kuypers, F. A., Shackleton, C., Brodbeck, U., and Stieger, S. (1990) J. Biol. Chem. 265, 18983-18987 [Abstract/Free Full Text]
  3. Deeg, M. A., Humphrey, D. R., Yang, S. H., Ferguson, T. R., Reinhold, V. N., and Rosenberry, T. L. (1992) J. Biol. Chem. 267, 18573-18580 [Abstract/Free Full Text]
  4. Doering, T. L., Pessin, M. S., Hart, G. W., Raben, D. M., and Englund, P. T. (1994) Biochem. J. 299, 741-746 [Medline] [Order article via Infotrieve]
  5. Edge, C. J., Rademacher, T. W., Wormald, M. R., Parekh, R. B., Butters, T. D., Wing, D. R., and Dwek, R. A. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 6338-6342 [Abstract]
  6. Fankhauser, C., Homans, S. W., Thomas-Oates, J. E., McConville, M. J., Desponds, C., Conzelmann, A., and Ferguson, M. A. J. (1993) J. Biol. Chem. 268, 26365-26374 [Abstract/Free Full Text]
  7. Ferguson, M. A. J. (1992a) in Lipid Modification of Proteins: A Practical Approach (Turner, A. J., and Hooper, N. M., eds) pp. 191-230, IRL Press, Oxford
  8. Ferguson, M. A. J. (1992b) Biochem. J. 284, 297-300 [Medline] [Order article via Infotrieve]
  9. Ferguson, M. A. J., Low, M. G., and Cross, G. A. M. (1985) J. Biol. Chem. 260, 14547-14555 [Abstract/Free Full Text]
  10. Ferguson, M. A. J., Homans, S. W., Dwek, R. A., and Rademacher, T. W. (1988) Science 239, 753-759 [Medline] [Order article via Infotrieve]
  11. Güther, M. L. S., Cardosa de Almeida, M. L., Yoshida, N., and Ferguson, M. A. J. (1992) J. Biol. Chem. 267, 6820-6828 [Abstract/Free Full Text]
  12. Hale, G., and Waldmann, H. (1994) Bone Marrow Transplantat. 13, 597-611 [Medline] [Order article via Infotrieve]
  13. Hale, G., Bright, S., Chumbley, G., Hoang, T., Metcalf, D., Munro, A. J., and Waldmann, H. (1983) Blood 62, 873-882 [Abstract]
  14. Hale, G., Xia, M.-Q., Tighe, H. P., Dyer, M. S. J., and Waldmann, H. (1990) Tissue Antigens 35, 118-12 [Medline] [Order article via Infotrieve]
  15. Homans, S. W., Ferguson, M. A. J., Dwek, R. A., Rademacher, T. W., Anand, R., and Williams, A. F. (1988) Nature 333, 269-272 [CrossRef][Medline] [Order article via Infotrieve]
  16. Kannagi, R., Stroup, R., Cochran, N. A., Urdal, D. L., Young, W. W., and Hakomori, S.-i. (1983) Cancer Res. 43, 4997-5005 [Abstract]
  17. Kay, R., Takel, F., and Humphries, R. K. (1990) J. Immunol. 145, 1952-1959 [Abstract/Free Full Text]
  18. Kay, R., Rosten, P. M., and Humphries, R. K. (1991) J. Immunol. 147, 1412-1416 [Abstract/Free Full Text]
  19. Kerwin, J. L., Tuininga, A. R., and Ericsson, L. H. (1994) J. Lipid Res. 35, 1102-1114 [Abstract]
  20. Kiarash, A., Boyd, B., and Lingwood, C. A. (1994) J. Biol. Chem. 269, 11138-11146 [Abstract/Free Full Text]
  21. Kirchhoff, C. (1994) Biol. Reprod. 50, 896-902 [Abstract]
  22. Kirchhoff, C., Krull, N., Pera, I., and Ivell, R. (1993) Mol. Reprod. Dev. 34, 8-15 [Medline] [Order article via Infotrieve]
  23. Kubota, H., Okazaki, H., Onuma, M., Kano, S., Hattori, M., and Minato, N. (1990) J. Immunol. 145, 3924-3931 [Abstract/Free Full Text]
  24. Masterson, W. J., Raper, J., Doering, T. L., Hart, G. W., and Englund, P. T. (1990) Cell 62, 73-80 [Medline] [Order article via Infotrieve]
  25. McConville, M. J., and Ferguson, M. A. J. (1993) Biochem. J. 294, 305-324 [Medline] [Order article via Infotrieve]
  26. Michell, R. H. (1975) Biochim. Biophys. Acta 415, 81-147 [Medline] [Order article via Infotrieve]
  27. Nyholm, P.-G., and Pascher, I. (1993) Biochemistry 32, 1225-1234 [Medline] [Order article via Infotrieve]
  28. Puoti, A., and Conzelmann, A. (1993) J. Biol. Chem. 268, 7215-7224 [Abstract/Free Full Text]
  29. Redman, C. A., Thomas-Oates, J. E., Ogata, S., Ikehara, Y., and Ferguson, M. A. J. (1994) Biochem. J. 302, 861-865 [Medline] [Order article via Infotrieve]
  30. Richier, P., Arpagaus, M., and Toutant, J.-P. (1992) Biochim. Biophys. Acta 1112, 83-88 [Medline] [Order article via Infotrieve]
  31. Riechmann, L., Clark, M. R., Waldmann, H., and Winter, G. (1988) Nature 332, 323-327 [CrossRef][Medline] [Order article via Infotrieve]
  32. Roberts, W. L., Myher, J. J., Kuksis, A., Low, M. G., and Rosenberry, T. L. (1988) J. Biol. Chem. 263, 18766-18775 [Abstract/Free Full Text]
  33. Schneider, P., and Ferguson, M. A. J. (1995) Methods Enzymol. 250, in press
  34. Schneider, P., Ralton, J. E., McConville, M. J., and Ferguson, M. A. J. (1993) Anal. Biochem. 210, 106-112 [CrossRef][Medline] [Order article via Infotrieve]
  35. Sherman, W. R., Ackerman, K. E., Bateman, R. H., Green, B. N., and Lewis, I. (1985) Biomed. Environ. Mass Spectrom. 12, 409-413
  36. Singh, N., Zoeller, R. A., Tykocinski, M. L., Lazarow, P. B., and Tartakoff, A. M. (1994) Mol. Cell Biol. 14, 21-31 [Abstract]
  37. Taguchi, R., Hamakawa, N., Harada-Nishida, M., Fukui, T., Nojima, K., and Izekawa, H. (1994) Biochemistry 33, 1017-1022 [Medline] [Order article via Infotrieve]
  38. Toutant, J.-P., Richards, M. K., Krall, J. A., and Rosenberry, T. L. (1990) Eur. J. Biochem. 187, 31-38 [Abstract]
  39. Valentin, H., Gelin, C., Colombel, L., Zoccola, D., Morizet, J., and Bernard, A. (1992) Transplantation 54, 97-104 [Medline] [Order article via Infotrieve]
  40. Volwerk, J. J., Shashidhar, M. S., Kuppe, A., and Griffith, O. H. (1990) Biochemistry 29, 8056-8062 [Medline] [Order article via Infotrieve]
  41. Walter, E. I., Roberts, W. L., Rosenberry, T. L., Ratnoff, W. D., and Medof, M. E. (1990) J. Immunol. 144, 1030-1036 [Abstract/Free Full Text]
  42. Wong, Y. W., and Low, M. G. (1994) Biochem. J. 301, 205-209 [Medline] [Order article via Infotrieve]
  43. Xia, M.-Q., Tone, M., Packman, L., Hale, G., and Waldmann, H. (1991) Eur. J. Immunol. 21, 1677-1684 [Medline] [Order article via Infotrieve]
  44. Xia, M.-Q., Hale, G., Lifely, M. R., Ferguson, M. A. J., Campbell, D., Packman, L., and Waldmann, H. (1993a) Biochem. J. 293, 633-640 [Medline] [Order article via Infotrieve]
  45. Xia, M.-Q., Hale, G., and Waldmann, H. (1993b) Mol. Immunol. 30, 1089-1096 [CrossRef][Medline] [Order article via Infotrieve]
  46. Yoshino, T., Watanabe, K., and Hakomori, S.-i. (1982) Biochemistry 21, 928-934 [Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.