Molecular characterization of Limulus polyphemus C-reactive protein. I. Subunit composition

zyxwvutsrqpo zyxwvuts zyxwvutsrqp zyxwv Eur. J. Biochem. 214, 99-110 (1993) 0 FEBS 1993 Molecular characterization of Limulus polyphemus C-reactive protein 11. Asparagine-linked oligosaccharides Supavadee AMATAYAKUL-CHANTLER’, Raymond A. DWEK’, Glenys A. TENNENT’, Mark B. PEPYSz and Thomas W. RADEMACHER’ Glycobiology Institute, Department of Biochemistry, University of Oxford, England zyxw ’ Immunological Medicine Unit, Department of Medicine, Royal Postgraduate Medical School, London, England (Received December 21, 1992Eebmary 22, 1993) - EJB 92 181712 The N-linked oligosaccharides of C-reactive protein (CRP) from the arachnid Limulus polyphemus, the horseshoe crab, were characterized after their release by hydrazinolysis, re-N-acetylation, and reduction with NaB3H,. High-voltage paper electrophoresis of the reduced oligosaccharides revealed only neutral species. Gel-permeation chromatography on Bio-Gel P4 yielded five fractions. The oligosaccharide fractions were further fractionated using high-voltage borate paper electrophoresis and Dionex BioLC ion-exchange chromatography. The oligosaccharides were structurally characterized by sequential exoglycosidase digestion, fragmentation by acetolysis and methylation analysis. Three major structures were found, of which two were the biantennary oligomannose type with compositions Man,GlcNAc, (B-l), Man,GlcNAc, (C-3) and one was the monoantennary structure Man,GlcNAc, (D-1). The biantennary oligomannose structures B-1 and C-3 contained the structural unit Mana6ManubR. This unusual arrangement of mannose linkages suggests a biosynthetic pathway in Limulus which differs from that reported in mammals, plants and the parasitic protozoa. C-reactive protein (CRP) is one of the most abundant proteins in the hemolymph of the arachnid Limulus polyphemus (the horseshoe crab) [l], and is of interest because it is phylogenetically the most ancient member of the stably conserved family of vertebrate plasma proteins known as pentraxins, which includes human C-reactive protein and serum amyloid P component [2,3]. Limulus CRP was isolated in a highly purified form in order to investigate its subunit composition [4], and we describe here the isolation of its N-linked oligosaccharides, their separation by high-resolution gel-permeation chromatography and their characterization by a combination of sequential exoglycosidase digestion, acetolysis fragmentation and methylation analysis. Little is currently known about the glycosylation characteristics of arachnid glycoproteins, but the present results show that Limulus CRP contains a series of novel biantennary oligomannose structures, suggesting a biosynthetic pathway in arachnids which differs from that reported in mammalian cells, plants and parasitic protozoa. MATERIALS AND METHODS Materials Hemolymph was pooled from about 20 L. polyphemus individuals and CRP was purified by phosphocholine affinity Correspondence to T. W. Rademacher, Dept of Immunology, University College London Medical School, Arthur Stanley House, 40-50 Tottenham Street, London, England W1 9PG Fax: +44 71 380 9357. Abbreviations. CRP, C-reactive protein; gu, glucose units (referring to chromatographic elution position from Bio-Gel P4; OT, tritiated reduced form of oligosaccharides. zy chromatography [ l , 41. /3-Mannosidase from Achatina fulica was a gift of Seikagaku Kogyo Co. Aspergillus phoenicis a( 1+2)-specific mannosidase, and a-mannosidase and p-Nacetylhexosaminidase from Canavalia ensifomis (jack bean) were purified [5]. Mana6(Manu3)Mana6(Mana3)Manprl GlcNAcp4GlcNAc abbreviated as Man’GlcNAc, (Oxford Glycosystems) and NaB3H, (5 - 15 Ci/mmol; New England Nuclear) were purchased. Tritium-labeled oligosacchariditols in solution were measured by scintillant counting (Beckman, LS 3801 counter) and detected on paper chromatograms and electrophoretograms (Packard Instruments Ltd or Berthold LB 2832 linear analyzer with a 30-cm or a 50-cm detector head, Lab-Impex Ltd). Exoglycosidase digestion Digestion of tritium-labeled reduced oligosaccharides (approximately 2 X lo5cpm) with exoglycosidases having defined specificities, were carried out under the following conditions: a(1+2) mannosidase (A. phoenicis), 20 pl 0.46 U/ ml in 0.1 M sodium acetate, pH 5.0 (1 U enzyme is defined by enzyme release of 1 pmol u-mannose from sodium borotritide-reduced mannan/min) ;jack bean p-N-acetylhexosaminidase, 20 p1 10 U/ml in 0.1 M citrate/phosphate, pH 4.0; jack bean a-mannosidase, 20 p1 50 U/ml in 0.1 M sodium acetate, pH 4.5. For jack bean a-mannosidase under specific conditions [6] where R+Manu6(Manu3)Man/34GlcNAcp4GlcNAc,, (OT, tritiated reduced form) but not Mana6(R+Mana3)Manp4GlcNAcp4GlcNAc, is susceptible. 5 X 10’ cpm oligosaccharide were incubated with 30 p1 10 U/ml jack bean a-mannosidase in 0.1 M sodium acetate, pH4.5; for /3-mannosidase (A. fulica), 10 pl 0.2 U/ml in 0.05 M so- 100 zyxwv zyxwvutsr zyxwvutsr zyxwvutsrqp dium citrate, pH 4.0 was used. For all the enzymes, unless specified, 1 U glycosidase was defined as the amount of enzyme that releases 1 pmol 4-nitrophenol from the respective 4-nitrophenylglycoside/min at 37 "C. All incubations were carried out for 18 h at 37°C under toluene, and were terminated by heating at 100°C for 2 min. Release, radiolabeling and isolation of asparagine-linked oligosaccharides Limulus CRP (7.5 mg) was dialyzed exhaustively against distilled water (4°C) and cryogenically dried over activated charcoal at - 196°C (<10 Pa). Approximately 300 pl fresh double vacuum-distilled anhydrous hydrazine (toluene/CaO, 25 "C, 1.4 kPa) was then added to the protein under an anhydrous argon atmosphere. The temperature of the reaction was raised from 30°C to 85°C at lO"C/h, then maintained at 85°C for a further 12 h. Hydrazine was removed by evaporation under reduced pressure at 25°C followed by repeated co-evaporation (five times) with anhydrous toluene (thiophene and carbonyl free). Re-N-acetylation was carried out with a fivefold molar excess of acetic anhydride (0.5 M) in saturated sodium bicarbonate for 10 min at O"C, then a second aliquot of acetic anhydride was added and further incubated for 50 min at room temperature. Sodium ions were removed by passage through Dowex AG-50X 12 (H' form). The salt-free hydrazinolysate was then evaporated to dryness at 27"C, redissolved in a minimum amount of water and applied to Whatman 3MM chromatography paper. After development for 48 h at 27 "C with butan-1-ol/ethanol/water (4 : 1:1, by vol.), the oligosaccharides were recovered from the first 5 cm from the origin of the paper by elution with water. The samples were evaporated to dryness, redissolved in a fivefold molar excess of 1 mM copper(I1) acetate over oligosaccharides, and incubated at 27°C for 45 min, before being desalted on tandem columns of Chelex-100 (Na') and Dowex AG-50 X 12 (H+). The salt-free oligosaccharides were taken to dryness, reapplied to Whatman 3MM chromatography paper, as described above, developed and eluted, and then passed through a 0.5-pm teflon filter (Millex SR, Millipore) and flash-evaporated to dryness (27 "C). The purified oligosaccharides were reduced with a fivefold molar excess of 6 mM NaB3H4(15 Ci/mmol) in 50 mM sodium hydroxide, adjusted to pH 11 with saturated boric acid at 30°C. After 4 h, an equal volume of 1 M NaB2H4 in buffered 0.05 M sodium hydroxide was added and the reaction was allowed to continue for a further 2 h. The reaction was terminated by dropwise addition of 1 M acetic acid, followed by removal of Na' through a Dowex AG-50 X 12 (H' form) column. The eluate was evaporated to dryness and the oligosaccharides were freed from boric acid by repeated evaporation (five times) with methanol. They were then dissolved in a minimum amount of distilled water and chromatographed on paper as described above. After 60 h, radioactivity was detected by radiochromatogram scanning and the radioactive oligosaccharides remaining at the origin of the paper were eluted with water. The oligosacchariditols were applied to Schleicher and Schuell (2043b 20 cm X 43 cm) paper and subjected to high-voltage electrophoresis in pyridine/acetic aciawater (3 : 1:387, by vol.) for 75 min at 80 Vcm-'. Radioactivity was detected by the linear analyzer. Neutral oligosacchariditols remaining at the point of application were eluted with water and passed through a column containing 0.1 ml each of Chelex-100 (Na' form), Dowex AG-50 X 12 (H' form), Dowex AG-3 X 4A (OH- form) and QAE-Sephadex A-25. The eluate was filtered through a 0.5pm teflon filter, evaporated to dryness and redissolved in 150 p1 water containing 0.5 mg of a mixture of isomaltooligosaccharides which were produced by partial acid hydrolysis of dextran. This solution was then applied to a highresolution gel-filtration system comprising two columns of Bio-Gel P4 (-400, 1.5 cmX 100 cm) in series. The columns were maintained at 55 "C and were incubated with water. The effluent was monitored by a Berthold HPLC radioactivityflow monitor (model LB 503, Lap-Impex) and an Erma refractive index monitor (model ERC 7510, HPLC Technology Ltd) prior to collection. Analog signals from these instruments were digitized using a Nelson Analytical ADC-interface and the digital values were collected and analyzed by computer (model 9836C, Hewlett-Packard). High-voltage borate electrophoresis Pooled oligosaccharides from Bio-Gel P4 were lyophilized, redissolved in a minimum amount of water and electrophoresed on Whatman 1 paper for 4.5-5 h at 8.5 kVm--' in 15 mM sodium tetraborate, pH 9.5, using a custom-built 1.2-m flat-bed high-voltage elecrophoresis unit (Locarte). Radioactive regions detected by the linear analyzer were eluted and evaporated to dryness. The samples were then dissolved, passed through Dowex AG-50 X 12 (H' form) and evaporated repeatedly (five times) with 1% (by vol.) acetic acid in methanol and once with water, to remove buffer salts. The samples were then evaporated to dryness, redissolved in 1 ml water and stored at -20°C prior to methylation analysis, sequential exoglycosidase analysis or acetolysis. Dionex BioLC ion-exchange chromatography An aliquot of each of the pooled oligosaccharides from Bio-Gel P4 was evaporated to dryness, redissolved in a minimum amount of water and applied to a Dionex BioLC system (Dionex UK Ltd). The Dionex Eluant Degas Module was used to sparge and pressurize the eluants with helium. The solutions were prepared by suitable dilution of a 12.5 M NaOH solution with glass-distilled water. A solution of either 0.1 M NaOH or 0.2M NaOH was used to separate neutral oligosaccharides by undergoing a 20-min isocratic elution. A column (4.6 mm X 250 mm) of Dionex Carbo Pac PA1 pellicular anion-exchange resin was fitted to the system with a Carbo Pac PA guard column (3 mmX 25 mm) and run at a flow rate of 1 ml/min at 30°C. Fractions of 250 p1 eluate were collected (Pharmacia Biosystems Ltd) and the radioactivity determined by liquid scintillation counting. Hydrazinolysis time-course analysis Approximately 0.61 mg Limulus CRP (dialyzed and lyophilized) was aliquoted into six hydrazinolysis tubes, followed by 0.29 mg of the internal standard GalP4GlcNAcp2Mana6 (GalP4 Glc NAcP2 Mana3) (GlcNAc/?4)Manp4GlcNAc~4(Fuca6)GlcNAc (unreduced, purified from sheep IgG). Tubes were then incubated in hydrazine at 85°C for 2 - 30 h before re-N-acetylation and isolation of oligosaccharides as above. The CRP-derived oligosaccharides were pooled separately from the internal standard, which eluted at 15.5 glucose units (gu), and the radioactivity determined. The ratio of the CRP peakhnternal standard was calculated for each time point. z zyxwvutsrqponm 101 1.4 I 0.0' 0 ' " " ' " zyxwvutsr zyxwvuts 1 zyx zyxwvutsr zyxwvuts zyxwvuts ' 10 20 incubation time (h) . " 30 10 Distance from origin (cm) Fig. 1. Hydrazinolysis time-course analysis of Limulus CRP. Limulus CRP was equally divided and added to six tubes containing a constant amount of an internal standard. To each tube, hydrazine was added and incubated as described in Materials and Methods. The incubation period of the glycoprotein in hydrazine at 85 "C varied over 2-30 h. The oligosaccharides liberated were pooled and the radioactively reduced as described in Materials and Methods. The CRP oligosaccharides were then separated from the internal standard by chromatography on Bio-Gel P4. The amount of sugar recovered from CRP was calculated from the amount of internal standard recovered. b zyxwvuts VO 1 201816 14 12 10 8 6 2 1 Partial acetolysis Partial acetolysis was carried out by modification of the procedure of Natsuka et al. [7]. Reduced oligosaccharides were dried and flash-evaporated with methanol (2 or 3 times). Pyridine and acetic anhydride (20 pl each) were added to the sample, mixed, centrifuged and left overnight at 20°C. The acetylated oligosaccharides were dried and flash evaporated with methanol (five times). The acetolysis reaction was performed by addition of 30 pl acetic anhydride/ acetic acidkoncentrated H,SO, (10: 1O:l) and incubation at 37°C for 6 h or more. Acid was removed by passing the solution through a column of Dowex AG3 X 4A (OH- form) and washing with 70% methanol. The eluant was evaporated to dryness and repeatedly evaporated with 70% methanol ( 5 X 200 pl). Methanol (100 pl) and ammonia (35%; 100 pl) were added to the acetylated oligosaccharides and the mixture was incubated overnight at 37°C. The deacetylation reaction was terminated by drying the mixture and passing through a column containing 250 p1 each of Chelex-100 (H' form) and Dowex AG50 X 12 (H' form). After filtration, the acetolysates were mixed with isomaltose oligomers and applied to Bio-Gel P4. Reducing terminal monosaccharide determination The identity of the reducing terminal monosaccharide was determined by radioelectrophoresis [8]. The reducing terminal monosaccharide, applied to a Whatman-1 paper sandwiched by two lanes containing monosaccharide standards, was electrophoresed for 5 h at 8.5 kVm-' in 15 mM sodium tetraborate, pH 9.5, as described above. Radioactive regions, detected by the linear analyzer for the unknown samples, were superimposed onto the standard to determine the reducing terminal monosaccharides. For absolute identification, equal amounts of the unknown and the standard monosaccharides were mixed together and re-run on highvoltage electrophoresis as described above. 300 500 700 900 1100 1300 Retention time (minutes) Fig. 2. High-voltage paper electrophoresis and Bio-Gel P4 chromatography of L. polyphemus CRP. (a) Tritium-labeled L. polyphemus C-reactive protein oligosaccharides were subjected to highvoltage paper electrophoresis (80 Vcm-') in pyridine/acetic acid water (3 : 1:387, by vol.) at pH 5.4.The arrows indicate the positions of ['Hllactitol (L), 6'(3')-~ialyl-[~H]lactitol(SL) and bromophenol blue (BPB) markers. (b) Oligosaccharides from pyridine electrophoresis were separated by high-resolution gel-filtration on Bio-Gel P4 as described in Materials and Methods. The number at the top represents the elution positions of dextran oligomers (number of glucose units). V,, void volume. The bars indicate the areas of peaks A-E that were pooled. The different traces are the P-4chromatograms of the six time-course samples (see Fig. 1). Methylation analysis Dextran-free oligosaccharides were subjected to methylation analysis according to a modification of the method of Ciucanu and Kerek [9]. Approximately 5 nmol (5 X lo6cpm) pure oligosaccharides were dissolved 50 p1 dimethyl sulfoxide and sonicated for 20 min. Then, in 50 p1 120 g/ml colloidal solution of NaOH in dimethyl sulfoxide was added to the solution, and the mixture incubated at room temperature, with stirring, for 30min. Three aliquots of methyl iodide (10 p1) were added and stirred for 10 min after each addition. After the last addition, the mixture was stirred for a further 10 min. The partially permethylated oligosaccharides were then extracted by addition of 300 pl chloroform and 1 ml 100 mg/ml sodium thiosulphate in water with vigorous mixing. After discarding the aqueous phase, the organic phase zyxwvut 102 zyxwvutsrqp zyxwvutsr zyxwvutsr ~~ (a1 zyxwvutsrq zyxwv zyxwvutsrqpon zyxwvutsrq zyxwvutsrqpon zyxwvutsrqponmlkjih Peak B 5-1 A PEAK A > r t c U 0 IJ a h 10 A 0 D-1 Peak D 0 (b) c-3 Peak C h 20 zyxwvutsrq I: 30 40 50 Distance from origin (cm) Fig. 3. Borate electrophoresis of L. polyphemus CRP neutral oligosaccharides. Pools from B-D Bio-Gel P4 (Fig. 2b) were subjected to high-voltage paper electrophoresis in 15 mM sodium borate, pH 9.5, as described in Materials and Methods. (a) Pool B ; (b) pool C ; (c) pool D. was extracted four times with 1 ml distilled water and the organic phase was evaporated to dryness under reduced pressure. To this, 100 p1 93% acetic acid in 0.25 M H,SO, was added to the sample and incubated at 80°C for a further 2.5 h. The reaction was terminated by passage of the mixture through a Dowex AG-3 X 4A (acetate form) column (500 pl) equilibrated in 50% methanol. The column was washed with 50% methanol and the eluant was dried and flash evaporated with 2 X 20 p1 toluene to obtain dry oligosaccharide samples. 200 p1 10 mg/ml solution of NaEVH, was added to the dry samples, sonicated for 5-10 min and incubated at 20°C for at least 2.5 h. The reaction was acidified with glacial acetic acid and borate ions were removed by flash evaporation with 2% acetic acid in methanol (5 X 300 pl). Then, 250 p1 acetic anhydride were added to the sample and incubated at 100°C for 2.5 h. Acetic anhydride was removed by evaporation under vacuum and the partially methylated alditol acetates were extracted by addition of 500 pl dichloromethane and 1 ml water. After thorough mixing of the above solution, the aqueous phase was discarded and the organic phase was concentrated to approximately 20 pl. Analysis of the partially methylated alditol acetates was performed on a Hewlett-Packard 5996C GLC-MS system fitted with on-column injection and flame-ionization detection. Separation was by capillary GLC on a bonded-phase CP-Sil8 CB column (0.32 mm X 25 m, Chrompak) with helium as the carrier gas. Direct on-column injection was employed with a temperature program of 90°C (held for 1 min), followed by a linear increase to 140°C at 30"C/min, then to 250°C at 5"C/min. Data were collected by selected ion monitoring, and identification of each partially methylated alditol acetate was based on the retention time and mass spectrum by comparison with synthetic reference compounds or published data. Preparation of Man,GlcNAc, and Man,GlcNAc, oligosaccharides from Man,GlcNAc, Mana6Mana6(Mana3)Man~4GlcNAc~4GlcNAc,, abbreviated as MaGGlcNAc, and Mana3Mana6Manp4Glc- PEAK C 10 20 30 40 Fraction number 50 Fig. 4. Ion-exchange chromatography of L. polyphemus CRP oligosaccharides by Dionex. Pools A-D from Bio-Gel P4 (Fig 2b) were subjected to a Dionex BioLC chromatography system. The oligosaccharides were separated by a 20-min isocratic elution in 200 mM NaOH at a flow rate of 1 mumin as described in Materials and Methods. Fractions of 250 pl eluant were collected and radioactivity associated with an aliquot of 10 p1 was determined for each column fraction for radioactivity in a liquid scintillant counter. The radioactivity in each fraction was plotted against time. (a) Pool A; (b) pool B ; (c) pool C; (d) pool D. NAcp4G1cNAcoT, abbreviated as Man,GlcNAc,, were derived from Mana6(Mana3)Mana6(Mana3)Manp4GlcNAcp4GlcNAcOT(Man,GlcNAc,) according to a modification of the method of Trimble et al. [lo]. Approximately 3 X lo6cpm Man,GlcNAc, was incubated at 37°C in 50 pl 10 mM sodium citrate, pH 4.5, containing 0.2 mM zinc acetate, with 0.5 U jack bean meal a-mannosidase. After 1.5 h, the reaction mixture was chilled to 0°C and a-mannosidase was removed by passing the reaction mixture through a column (1 cm X 2 cm) of Whatman P11 phosphocellulose equilibrated with 0.05 M sodium citrate, pH 5.5. The eluate was concentrated to 1.O ml by flash evaporation and desalted by pass- z zyxw zyx 103 Table 1. Methylation analysis of oligosaccharides B-1, C-3 and D-1. Oligosaccharides B-1, C-3 and D-1 were released by hydrazinolysis and radioactively reduced as described (Materials and Methods). Following separation of the oligosaccharides by Bio-Gel P4 gel-permeation chromatography and borate electrophoresis, individual fractions were separately pooled (see Figs 2b and 3 ) and methylation analysis was performed as described (Materials and Methods). Molar ratios are expressed relatively to 2,4-di-O-methyl (1,3,5,6-tetra-O-acetyl) mannitol. Methylated Linkage Oligosaccharides zy zyxwv moVmol B-1 Mannitol 2,3,4,6-Tetra-O-methyl(1,5-di-O-acetyl) 2.4 0.7 2,4,6-Tri-O-methyl(1,3,S-tri-O-acetyl) 2,3,4-Tri-O-methyl(1,5,6-tri-O-acetyl) 2,4-Di-O-methyl(1,3,5,6-tetra-O-acetyl) terminal 2 3 6 3.6 2-(N-Methylacetamido)-2-deoxyglucitol 3,6-Di-O-methyl(l,4,5-tri-O-acetyl) 1,3,6-Tri-O-methy1(4,S-di-O-acetyl) 4 terminal 0.9 trace 3,4,6-Tri-O-methy1(1,2,S-tri-O-acetyl) - 0.9 1.o c-3 D-1 3.1 1.o - 1.4 1.o 0.6 trace - 0.1 0.6 0.2 0.9 trace zyxwvutsr Fraction number Fig. 5. Elution profiles of oligosaccharide A-1 on Bio-Gel P4 after treatment with various exoglycosidases and partial acetolysis. (a) Bio-Gel P4 of oligosaccharide A-1 (Fig. 4) after treatment with A. phoenicis a-mannosidase; (b) elution profile of oligosaccharide A-1 after partial acetolysis; (c) elution profile of oligosaccharide A-I acetolysate (from Fig. 5b) after treatment with jack bean a-mannosidase ; and (d) elution profile of oligosaccharide A-I treated with A. phoenicis a-mannosidase, then acetolysed. See Materials and Methods for the experimental procedures. The numbers at the top represents the elution positions of dextran oligomers (number of glucose units). The bold arrow indicates the starting elution position. ing through a tandem column of 2.0ml each of Dowex AG3 X4A (OH- form) and Dowex AG50 X 12 (H' form). The salt-free oligosaccharides were then subjected to Dionex BioLC chromatography. The products were separated by a 20-min isocratic elution in 100 mM NaOH at a flow rate of 1.0 mumin. Fractions of 250 p1 were collected and an aliquot of 20 pl was counted for radioactivity. The radioactive areas were pooled separately, neutralized with 1 M acetic acid to pH 5.5, desalted as described above and chromatographed on Bio-Gel P4. The two major peaks were Mana6Mana6(Mana3)Manp4GlcNAc~4GlcNAc, and Mana3Mana6Man~4GlcNAc~4GlcNAc,, The Mana6Mana6(Mana3)Manp4GlcNAcp4GlcNAcoT oligosaccharide standard made above was then mixed with an equivalent amount of CRP C-3, while the Mana3Mana6Man~4GlcNAc~4GlcNAco.oligosaccharide standard was mixed with CRP D-1. Both mixtures were chromatographed on a Dionex BioLC system and eluted with 100 mM NaOH 104 zyxwvutsrqp zyxwvutsrqponm zyxwvutsrqpon zyxwvutsrqponm zyxwvutsrqponm zyxwvutsrqpo 1816 14 12 10 8 ,tlttittO 1 i t 6 t 4 i i t B C D E F G A 2' 1 1 1 1 1 1 1 1 I 9 8 7 6 5 1 a a J H zyxwvutsrq b c I 9 0 7 6 5 4 I t t1.Jt t i 300 400 500 Retention time (minutes) C 20 40 60 Distance from origin (cm) Fig. 7. Borate electrophoresis of the reducing terminal residue of oligosaccharides A-1, B-1, C-3 and D-1. Oligosaccharides A-1 (Fig. 4), B-1 (Fig. 3), C-3 (Fig. 3) and D-1 (Fig. 3) were subjected to acetolysis and to various exoglycosidases. After the oligosaccharides were digested to 2.5 gu, they were subjected to high-voltage paper electrophoresis in 15 mM sodium borate, pH 9.5, as described in Materials and Methods. (a) shows the migrated distance of the oligosacchariditol standards. A = 2-deoxyribitol, B = N-acetylmannosaminitol, C = N-acetylglucosaminitol, D = xylitol, E = glucitol, F = mannitol, G = fucitol and H = galactitol. (b) shows the migrated distance of the 2.5-gu obtained from oligosaccharides A-l, B-1. C-3 and D-1. drazinolysis reaction. Liberation of the oligosaccharides reached 50% by approximately 8 h. The yield of the oligosaccharides released from Limulus CRP was compared to that of the internal standard and approached 1 chaidsubunit at approximately around 28 h of the reaction. When the hydrazinolysis time-course of the human serum amyloid P component (SAP, a phylogenetically related protein) was studied, liberation of the SAP oligosaccharides reached completion within 12- 13 h of hydrazinolysis (unpublished results). Studies on other glycoproteins such as transferrin, human IgG and Erythrina cristagalli lectin have also previously shown that they too are completely liberated after 12 h (unpublished results). Fig. 2b shows the high-resolution gel-permeation P4 profile of the released oligosaccharides at different time points ranging over 2-30 h. No selective release of oligosaccharides is evident and the proportions of released oligosaccharides at 2 h and 28 h are essentially identical. The reason for the extended amount of time needed to liberate oligosaccharides from Limulus CRP by hydrazinolysis is not understood at present. zyxwvutsrq 600 Fig. 6. Elution profiles of oligosaccharide B-1 on Bio-Gel P4 after treatment with various exoglycosidases and partial acetolysis. Oligosaccharide B-I (Fig. 3) was incubated with A. phoenicis amannosidase and applied onto Bio-Gel P4 (a). The digested peak was then subjected to acetolysis and Bio-Gel P4 (b). The acetolysate eluting at 6.4 gu (indicated in the bar) was then treated with jack bean a-mannosidase and applied onto Bio-Gel P4 (c) and subsequently treated with A. fulica j?-mannosidase (d). Finally, the digested peak, now eluting at 4.5 gu, was treated with jack bean p-Nacetylhexosaminidase and subjected to Bio-Gel P4 (e). The numbers at the top represents the elution positions of dextran oligomers (number of glucose units). as described above. The radioactivity of the fractions was determined, plotted against time and compared to those of CRP C-3 and CRP D-1. Oligosaccharides isolated from Limulus C-reactive protein Pyridine acetate high-voltage electrophoresis of the oligosaccharides obtained by treatment of Limulus CRP with hydrazine, followed by re-N-acetylation and reduction with NaB3H,, demonstrated that Limulus CRP oligosaccharides comigrate with lactose and are therefore neutral (Fig. 2a). The neutral fraction was eluted from paper with distilled water and subjected to gel-filtration chromatography on BioGel P4. This procedure, in which oligosaccharides are sepa- zyxwvutsrqpo RESULTS Hydrazinolysis time-course analysis of Limulus CRP Fig. 1 shows that the quantity of oligosaccharides released from Limulus CRP increased with the time of the hy- z zyxwvutsrqponm zyxwvutsrqp zyxwvutsrqp zyxwvutsrqpo 105 18 16 14 12 10 8 7 I t t i t l t t i l i i i t 6 t 5 t 4 i 3 T zyxwvuts 8 7 6 I i i b I I 5 I zyxwvutsr zyxwvutsrqpo 300 400 500 600 Retention time (minutes) 6 5 4 Fig. 8. Bio-Gel P4 profile of oligosaccharideB-1 after partial acetolysis and A. phoenicis a-mannosidase. Oligosaccharide B-1 (Fig. 3) was subjected to acetolysis and applied onto Bio-Gel P4 (a). The acetolysate (eluting at 7.4 gu) was digested with A. phoenicis a-mannosidase according to the procedures described in Materials and Methods. (b) shows the Bio-Gel P4 profile of the 7.4 gu product of (bar in Fig. 8a) acetolysate after treatment with A. phoenicis amannosidase. The arrow in bold-face type indicates the elution position of the starting material. zyxwvutsrq rated by their differences in hydrodynamic volumes, yielded five components (Fig. 2b) at all time points studied. Peak A (2.9%, Fig. 2b) eluted at 9.8 gu compared to internal isomalto-oligosaccharide standards. Oligosaccharides in peak B (15.7%, Fig. 2b) eluted at 8.9 gu and those in peak C (15.0%) at 7.9 gu. The majority of the oligosaccharides (i.e. 64.2%) eluted in peak D at 6.9 gu (Fig. 2b) followed by a smaller shoulder peak, E, eluting at approximately 6.1 gu (2.3 %). For further fractionation, oligosaccharides were pooled as shown in Fig. 2b. Pools B, C and D were subjected to high-voltage paper electrophoresis in borate buffer at pH 9.5 (Fig. 2b). Pool B from Bio-Gel P4 yielded component B-1 (Fig. 3a) and a minor component eluting at the same distance as peak C-3 (see Fig. 3b). Upon rechromatography on Bio-Gel P4, a single peak at 8.9gu was obtained from compound B-1 (data not shown). Pool C contained a major peak, C-3, and minor peaks, C-1, C-2 and C-4 (Fig. 3b). Peaks C-1 and C-2 are overlapping peaks from component D-1 and B-1, respectively (see Fig. 3a,c). When peak C-3 was rechromatographed on Bio-Gel P4, a single peak eluting at 7.9 gu was observed (see Fig. 9a). Pool D from Bio-Gel P4 yielded a major component, D-I and a minor component D-2 which migrate to the same position as peak C-3 (Fig. 3c). Peak D-1 was shown to be a single peak eluting at 6.9 gu when reapplied onto Bio-Gel P4. Pool E was not analyzed due to insufficient material. Pools A-D were also chromatographed on a Dionex BioLC system. Pool A yielded a major component, A-1 and a minor component (A-0) (Fig. 4a). The latter component was not analyzed further. After elution of compound A-1 I i t c t e , T 300 LOO 500 600 Retention time (minutes) Fig. 9. Structural analysis of oligosaccharide C-3 by partial acetolysis, exoglycosidase digestion and Bio-Gel P4. Oligosaccharide C-3 (Fig. 3) was treated with A. phoenicis a-mannosidase and applied onto Bio-Gel P4 (a). Acetolysis of oligosaccharide C-3 fragmented it to a smaller oligosaccharide eluting on Bio-Gel P4 at 6.4 gu (b). The 6.4 gu oligosaccharide was then sequentially treated with (c) jack bean a-mannosidase, (d) A. fufica P-mannosidase and (e) jack bean P-N-acetylhexosaminidasefollowed by Bio-Gel P4 after every digestion as described in Materials and Methods. The arrow in bold-face type indicates the elution position of the starting material. from the column and rechromatography on Bio-Gel P4, a single peak at 9.8 gu was obtained (data not shown). Figs. 4b, c and d shows that pools B, C and D resolved into peaks with similar patterns to the electrophoretograms when purified by borate electrophoresis (see Fig. 3). Pool B contained one major peak and two minor peaks. The minor peaks are probably overlapping peaks from pools A and C (Fig. 4b). Pool C also contained a major peak and two minor peaks. One of the minor peaks is an overlapping peak from pool D while the other is probably peak C-4 seen in Fig. 3b (Fig. 4c). In zyxw 106 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA /:Bl’l”,’f,’PI’P,i? i A, 26000. 2 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA 1 a 20000 16000 12000 8000 zyxwvu 1000 0 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHG + i I t 5 V Y .-). > .-c V m .-0 4- 73 m a zyxwv t zyxwvutsrq zyxwvutsrqp zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA LOO 500 t Relent ion ti me ( m i n u t e s ) Fig. 10. Structural analysis of oligosaccharide D-1 by partial acetolysis, exoglycosidase digestion and Bio-Gel P4. Oligosaccharide D-1 (Fig. 3) was treated with A. phoenicis a-mannosidase and applied onto Bio-Gel P4 (a). Acetolysis of oligosaccharide D-1 fragmented it to a smaller oligosaccharide eluting on Bio-Gel P4 at 5.5 gu (b). The 5.5 gu oligosaccharide was then sequentially treated with different exoglycosidases followed by Bio-Gel P4; (c) A. fulica P-mannosidase and (d) jack bean P-N-acetylhexosaminidase. The arrow in bold-face type indicates the elution position of the starting material. 300 pool D, one major peak and a very minor peak was resolved after the pool had been subjected to chromatography on a Dionex BioLC system (Fig. 4d). Methylation analysis of oligosaccharides B-1, C-3 and D-1 Table 1 shows that the three major oligosaccharides (i.e. B-1, C-3 and D-l), consists of only mannose and N-acetyl- 200 300 Loo 500 Retention time (minutes) Fig. 11. Ion-exchange and gel-filtration chromatographies of Man,GlcNAc, after treatment with jack bean a-mannosidase. (a) Man,GlcNAc, was digested with jack bean a-mannosidase as described in Materials and Methods. After the reaction was terminated, the sugars were applied onto a Dionex BioLC chromatography. The oligosaccharides were separated by a 20-min isocratic elution in 100 mM NaOH at a flow rate of 1.0mumin. Fractions of 250 p1 were collected and the radioactivity determined as described above. Compounds B and C were pooled separately and applied onto a BioGel P4 chromatography (b and c, respectively). The arrows at the top of the figure represents the elution positions of dextran oligomers. oligosaccharide C-3, methylation analysis suggests that the oligosaccharide contains three terminal mannoses, one 6linked mannose, one 3,6-linked mannose and one 4-linked glucosamine residues. This suggests that the oligosaccharides N-acetylglucosamine and a reducing terminal N-acetylglucoare either of the oligomannose andlor of the hybrid type. saminitol residue. Note that only trace amounts of the parMethylation analysis of oligosaccharide B-1 suggests that the tially methylated alditol acetate, corresponding to the reducoligosaccharide contains two terminal mannose residues, a 6- ing terminal N-acetylglucosaminitol residue, were detected linked mannose residue, a 2-linked mannose, a 3,6-linked due to its instability. mannose residue, a 4-linked N-acetylglucosamine residue Methylation analysis of peak D-1 suggests that the and a reducing terminal N-acetylglucosaminitol residue. For zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCB oligosaccharide contains, in addition to a standard N,Nf-di- zyxwvutsrqponml zyxwvutsrqponm 107 12000 1600 a 10000 8000 6000 4000 5 a-mannose residues (data not shown). Partial acetolysis of peak A-1 resulted in a major peak eluting at 7.5 gu, a minor peak eluting at 8.9 gu and some starting material (Fig. 5b). However, acetolysis of peak A-1 after A. phoenicis a(1+2)specific mannosidase digestion resulted in a major peak, a minor peak and some starting material eluted at 6.5 gu, 7.9 gu and 8.9 gu, respectively (Fig. 5d). Since acetolysis cleaves an oligosaccharide only at a1-6 linked mannose residues, this suggests that the a(1-2) mannose residue is linked to A-1 via the mannose residue linked al+3 to the p-mannose residue. Treatment of all the components generated by acetolysis with jack bean a-mannosidase resulted in a single peak eluting at 5.5 gu on Bio-Gel P4 (Fig. 5a). Further treatment of the 5.5 gu oligosaccharide with A. fulica p-mannosidase and, subsequently, with jack bean P-N-acetylhexosaminidase resulted in a peak eluting at 2.5 gu due to the loss of a p-mannose and a p-N-acetylhexosamine residue (data not shown). zyxwvutsrqponm - zyxwvutsr zyxwvutsrq 2000 Ea 0 v .-% .-> 0 c 0 a 12000 Oligosaccharide B-1 0 .- 2 Treatment of oligosaccharide B-1 (8.9 gu) with A. phoenicis 4 1 - 9 2 ) mannosidase resulted in the loss of one a1-2 linked mannose (see Fig. 6a). Partial acetolysis of the di8000 gested peak, now at 7.9, gu yielded a smaller oligosaccharide eluting at 6.4 gu (Fig. 6b) indicating the loss of two mannose 6000 residues, as well as starting material. When this 6.4-gu oligosaccharide was treated with jack bean a-mannosidase, 4000 the oligosaccharide shifted from 6.4 gu to 5.5 gu due to loss of another a-linked mannose residue (Fig. 6c). Subsequent treatment of this 5.5-gu oligosaccharide with A. fulica p2000 mannosidase and jack bean /I-N-acetylglucosaminidaseresulted in a peak at 2.5 gu (loss of one p-mannose residue 0 (4.5 gu) and a P-N-acetylglucosamine residue (2.5 gu), re4 6 8 10 12 spectively; Fig. 6d,e). The radioactivity eluting at 2.5 gu was analyzed by high-voltage borate electrophoresis and conRetention time (rnin) firmed to be p-N-acetylglucosaminitol(Fig. 7a, b). Fig.12. HPLC analysis of the oligosaccharides from CRP verPartial acetolysis of oligosaccharide B-1 (8.9 gu) genersus oligosaccharide standards. Aliquots of CRP C-3 (a) and CRP D-1 (b) were applied onto a Dionex BioLC system fitted with a ated a peak eluting at 7.6 gu which resulted from loss of one Dionex Carbo Pac PA1 pellicular anion-exchange resin running in to two a-linked mannose residues (Fig. 8a). Treatment of the 100 mM NaOH at a flow rate of 1.0 mumin at 30°C as described in acetolysate with A. phoenicis 41-2) mannosidase, resulted in the loss of one a1-2 linked mannose residue and moved Materials and Methods. In addition, the Mana6Mana6(Mana3)Manp4GlcNAc/34GlcNAcoTand the Mana3Mana6Man/l4GlcNAcp4- the peak to 6.4 gu (Fig. 8b). Sequential digestion of this GlcNAc,, standard sugars were mixed in equal amounts with CRP 6.4 gu species (as described above for oligosaccharide B-l), C-3 (a) and CRP D-1 (b), respectively. The mixtures of equal radio- resulted in shifts of the oligosaccharide elution on Bio-Gel activity were applied to Dionex BioLC under identical conditions as P4 in an identical manner. 10000 zyxwvutsrqponmlkj zyxwvutsrqpo above and compared. Dotted line = oligosaccharides from CRP, solid line = CRP oligosaccharide plus standard. Oligosaccharide C-3 acetylchitobiosyl core, a 3-linked mannose residue and a 6linked mannose residue. A small amount of 3,6-linked mannose was also found, but both chromatography of D-1 on borate HVE and Dionex (Figs 3c, 4d, 12b) show only a single species present. zyxwvut zyxwvutsrq Exoglycosidase digestion and partial acetolysis of Limulus CRP oligosaccharides Oligosaccharide A-1 The terminal mannose residues of oligosaccharide C-3 were not removed by treatment with A. phoenicis 41-2) mannosidase (Fig. 9a). However, acetolysis fragmented the 7.9 gu into a smaller oligosaccharide eluting at 6.4 gu by removing two a-mannose residues (Fig. 9b). The major product eluting at 6.4 gu showed identical exoglycosidase digestion patterns to those obtained from oligosaccharide B-1, as listed above (Fig. 9c-e). One terminal mannose residue of oligosaccharide A-1 was removed by treatment with A. phoenicis a(l+2)-specific mannosidase (Fig. 5a). Treatment of oligosaccharide A-1 with jack bean a-mannosidase, however, resulted in a product which eluted at 5.5 gu on Bio-Gel P4, indicating the loss of Oligosaccharide D-1 Peak D-1 was not digested by A. phoenicis 41-2) mannosidase and therefore does not contain terminal a1-2 mannose residues (Fig. 10a). Partial acetolysis of peak D-1 generated a fragment eluting at 5.5 gu as well as starting material (Fig. lob). After treatment of the 5.5 gu peak with A. 108 zyxwvutsrqp zyx Relative Mol [Mana3(6)I2Mana Mana2Mana (“A) \‘ Man p 4GlcNAcp4GlcNAc oligosaccharide A-1 2.6 \6 Man p 4GlcNAcp4GlcNAc oligosaccharide B-1 16.0 /3 ManaGMana Mana2Mana /3 Mana6Mana zyxw zyxwvutsr zyxwvut “Man p 4GlcNAcp4GlcNAc Mana /3 Man &Man a6Man PGlcNAcp4GlcNAc oligosaccharide C-3 18.6 oligosaccharide D-1 57.3 Fig. 13. Proposed structures of oligosaccharides A-1, B-1, C-3 and D-1. The structures of oligosaccharides A-1, B-I, C-3 and D-1 was assessed from the data after treatment with various exoglycosidases, GC-MS and acetolysis. The molar proportion of each structure is summarized. zyxwvut fulica P-mannosidase, the oligosaccharide eluted at 4.5 gu (Fig. IOc), due to loss of one P-mannose residue. When the 4.5-gu structure was digested with jack bean P-N-acetylhexosaminidase, one P-N-acetylglucosamine residue was liberated, moving the oligosaccharide to 2.5 gu (Fig. 1Od). This 2.5-gu oligosaccharide was later confirmed to be N acetylglucosaminitol (see Fig. 7b). charides Limulus CRP C-3 and CRP D-1 when run alone (Fig. 12a,b). A summary of the proposed structure of the oligosaccharides A-I, B-1, C-3 and D-1 is shown in Fig. 13. DISCUSSION Limulus CRP was found to contain three major N-linked oligosaccharides, all of which contained only mannose and and Mana3Mana6Man~4GlcNAcp4GlcNAc,, N-acetylglucosamine residues (Fig. 13). The most abundant oligosaccharides prepared from Man,GlcNAc, species (57 %) was a linear Mana3Mana6Manp4GlcNAcp4compared with Limulus CRP C-3 and D-1 GlcNAc structure. Oligosaccharides containing Fig. l l a shows an anion exchange chromatogram of Man,GlcNAc, structures have been reported previously in Mana6(Mana3)Mana6(Mana3)Man~4GlcNAc~4GlcNAc,, invertebrates, but have always been branched structures. Glyafter treatment with jack bean meal a-mannosidase for 1.5 h coproteins containing these structures include hemocyanin at 37°C. Four components were observed (Fig. l l a ) with from the spiny lobster, Palinurus interruptus [ l l ] and from peaks B and C representing as much as 85% of the total the gastropods Helix pomatia [12] and Lymnaea stagnalis radioactivity. Upon rechromatography on Bio-Gel P4, a [13, 141 membrane glycoproteins from Drosophila [151, lysingle peak at 6.9 gu was obtained from compound B (see sosomal enzymes from Tetrahymena pyriformis [ 161 and neFig. llb). The majority of the radioactivity (56%) eluted at ural glycoprotein of the marine mollusc Aplysia californica peak C was found to be a single peak eluting at 7.9 gu on ~171. Bio-Gel P4 (Fig. llc). Pools A (5%) and D (10%) eluted at The biantennary Man,GlcNAc, (C-3) and Man,GlcNAc, 6.1 gu and 8.9 gu, respectively (data not shown). According (B-1) oligosaccharides were found to differ from each other to the hydrodynamic volume on Bio-Gel P4, the oligosaccha- by the presence of a Mana2 residue attached to the Mana3 rides in peaks A-D are most likely to be Man,GlcNAc,, branch in B-1. Both C-3 and B-1 were found to contain a Man,GlcNAc,, Man,GlcNAc, and the starting material, Mana6 residue linked to the Mana6 ‘arm’.Structures similar Man,GlcNAc,. to C-3 and B-1 have recently been reported to occur in Gp63, According to Trimble et al. [lo], the Man,GlcNAc, struc- a major surface glycoprotein, from the parasite Leishmania ture C is Mana6Mana6(Mana3)Man~4GlcNAc~4GlcNAcOTmexicana amazonensis [5]. However, Gp63 contains a and the Man,GlcNAc, structure B is Mana3Mana6M- Mana3 residue linked to the Mana6 ‘arm’. an/l4GlcNAc~4GlcNAc,,. Standard oligosaccharide B was The absence of complex-type and hybrid-type oligosacmixed with Limulus CRP D-1 and standard oligosaccharide charides could be due to the absence of GlcNAc-transferase C was mixed with Limulus CRP C-3, in both cases at molar I in Limulus or to the inaccessibility of the glycosylation ratios of 1 : 1. The mixtures were then chromatographed on a sites of CRP to the enzyme. There is only limited data on Dionex BioLC system (Fig. 12), and in each case a single oligosaccharide structures available from other arthropods. peak was observed eluting at the same position as oligosac- The crustacean, P: interruptus, contains the oligosaccharide, Mana6Mana6(Mana3)Manp4GlcNAcp4G1cNAcoT zyx zyxwv zyx zyxw zyxwvu zyxwv 109 Mana6(GlcNAcp2Mana3)Man~4GlcNAc~4GlcNAc, suggestive of the presence of a GlcNAc-transferase I activity [11]. However, structures of oligosaccharides from Drosophilu and Aedes would suggest the absence of GlcNAc-transferase I in these species [15, 18, 191. Recently, complex-type oligosaccharides have been observed in the lepidopteran insect (Spodopteru frugiperdu) infected with a recombinant baculovirus containing the entire human plasminogen cDNA [20]. Therefore, enzymes such as mannosidases, galactosyltransferases, N-acetylhexosaminyltransferase, and sialyltransferases can be expressed in certain arthropods. The Man,GlcNAc,, Man,GlcNAc,, MaaGlcNAc, and Man,GlcNAc, oligomannose structures described here are unusual with respect to the oligomannose structures typically isolated from mammalian glycoproteins. In the common processing pathway, all 2-linked mannose residues are removed from a Man,GlcNAc, precursor to produce a Man,GlcNAc, structure which then loses two further mannosyl residues only after addition of a GlcNAc residue to the a(l-3) arm. The GlcNAc-transferase-I-dependent Golgi mannosidase I1 that removes the final two mannosyl residues is highly specific for GlcNAc,Man,GlcNAc, and has no activity against Man,GlcNAc, [21, 221. Thus, among the oligomannose structures usually observed, the smallest is Man,GlcNAc, and all have a branched (3,6-linked) substituted mannosy1 residue. At present it is not known whether N-linked oligosaccharides from Limulus are synthesized via a glucosylated pyrophosph~ryldolichol oligosaccharide precursor. However, other arthropods such as mosquitoes and fruit flies, are known to synthesize glucose-containing lipid-linked oligosaccharides with properties identical to that of Glc,Man,GlcNAc,-P-P-dolichol [ 19, 231. In contrast, in Leishmania, the phosphoryldolichol oligomannose precursor is non-glucosylated Man,GlcNAc,-P-P-dolichol [24]. Trimble et al. [lo] found that limited a-mannosidase digestion of Man,GlcNAc, resulted in two compounds, Mana6Mana6(Mana3)Manp4 GlcNAc~4GlcNAco, and Mana3Mana6Man~4GlcNAc~4G1cNAcoT. These two structures were found to co-elute with the Limulus CRP-derived oligosaccharides Man,GlcNAc, (C-3) and Man,GlcNAc, (D-l), respectively. Since the largest oligosaccharide found in Limulus CRP is a Man,GlcNAc, structure, the presence of an a-mannosidase with a specificity similar to the jack bean a-mannosidase activity and an a(1-2) mannosidase-like activity are all that is needed to generate the structures on Limulus CRP. Future studies on more arachnid glycoproteins and analysis of the glycosidases present in Limulus will be necessary to confirm the biosynthetic pathway for oligosaccharide processing. We thank Dr Ian Manger and Dr Jerry Thomas for their advice on the GC-MS of CRP oligosaccharides. The Oxford Glycobiology Institute is supported by Monsanto. This work was supported in part by Medical Research Council Programme grant G7900510 to M. B. P. REFERENCES 1. Robey, F. A. & Liu, T.-Y. (1981) Limulin: a C-reactive protein from Limulus polyphemus, J. Biol. Chem. 256, 969-975. 2. Osmand, A. P., Friedenson, B., Gewurz, H., Painter, R. H., Hofmann, T. & Shelton, E. (1977) Characterisation of C-reactive protein and the complement subcomponent Clt as homologous proteins displaying cyclic pentameric symmetry (pentraxins), Proc. Natl Acad. Sci. USA 74. 739-743. 3. Pepys, M. B. & Baltz, M. L. 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